Cleavable surfactants

ABSTRACT

The invention provides surfactant compounds of formulas I-IX, which can be used in methods for aiding the solubilization, digestion, preparation, analysis, and/or characterization of biological material, for example, proteins or cell membranes. The compounds can also aid in the recovery of peptides generated during protein digestion, particularly for in-gel digestion protocol. Additionally, the compounds can improve enzymatic protein deglycosylation without interfering with downstream sample preparation steps and mass spectrometric analysis. The compounds can be specifically useful as digestion aids that can be decomposed by an acid, by heat, or a combination thereof. Decomposition of the surfactants allows for facile separation from isolated samples, and/or allows for analysis of the sample without interfering with the sensitivity of various analytical techniques.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 60/979,316, filed Oct. 11, 2007,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The preparation of protein samples for analysis, for example, massspectrometry analysis, typically includes three main steps:solubilization, digestion, and peptide recovery. Current methods andreagents applied to one step are rarely compatible with the followingstep. For example, solubilizing agents such as surfactants (e.g., sodiumdodecyl sulfate (SDS)) or denaturants (e.g., acetonitrile, urea, orguanidine) typically inhibit the proteases, such as trypsin, that areused in the digestion that follows solubilization. Even when used inconcentrations that can be tolerable for trypsin activity, the presenceof these surfactants or denaturants interfere with subsequent analyses,such as liquid chromatography or mass spectrometric analysis.Accordingly, removal of the surfactants and organic solvents istypically required before conducting further analysis on a sample (e.g.,proteins or peptides). The manipulations required for removal of thesereagents complicates the sample preparation process and often leads toloss of sample material.

The digestion step frequently presents a major challenge in proteinsample preparation. A typical protein digestion with trypsin requiresovernight incubation to reach completion. Even after overnightincubation, some proteins that are resistant to digestion, such asmembrane proteins, can remain intact, thus requiring extraordinaryconditions to achieve satisfactory digestion. Current methods employedin an attempt to overcome these limitations and to speed the digestionprocess include the use of organic solvents (e.g., acetonitrile),elevated temperatures, denaturants (e.g., urea), and/or detergents(e.g., SDS) to improve protein solubilization and protein denaturation,thus improving digestion. However, these alternative methods andadditives often result in incomplete cleavage and low reproducibility,limiting their utility. The use of these reagents also leads toinhibition of trypsin activity, interference with HPLC separation, andsuppression of peptide detection in mass spectrometry.

In-gel protein digestion brings specific challenges to protein samplepreparation. Success of in-gel digestion relies not only on efficientprotein digestion but also on efficient post-digestion peptideextraction from the gel. Peptide extraction from the gel can be lengthyand laborious and it is often only moderately efficient in terms ofpeptide recovery. Recovered peptides are generally limited to the sizeof about 2,500 Da. Longer peptides are largely trapped in the gel. See“In-gel digestion with endoproteinase. Lys-C”, Y. Wada, M. Kadoya, J. ofMass Spectrom. 2003; 38: 117-118. Recovery of peptides with increasedhydrophobicity is also impacted.

Other procedures related to protein sample preparation include analysisof post-translational protein modifications. About 60% of all humanproteins are glycosylated. Glycosylation was shown to play importantrole in many key cellular mechanisms. To analyze glycosylation, a glycanshould be separated from a protein. This removal, referred to asdeglycosylation, is performed by using glycosidases. Deglycosylation isfrequently a time-consuming process. Reagents such as sodium dodecylsulfate (SDS) can dramatically improve deglycosylation, potentially byproviding better access to glycan attachment sites for the glycosidases.However, SDS interferes with downstream sample preparation steps, massspectrometric analysis, and HPLC analysis.

Accordingly, there is a need for improved methods for protein samplepreparation. There is also a need for methods or reagents that benefitone or more of the three major protein preparation steps:solubilization, digestion, and peptide recovery, in order to streamlinethe protein sample preparation process. Preferably, these methods orreagents would not lead to the inhibition of protease activity, andwould not interfere with isolation and/or characterization techniques.There is a particular need to streamline in-gel digestion protocols andto improve recovery of peptides from gels. Finally, there is a need forimproved methods of protein deglycosylation that do not interfere withdownstream sample preparation and mass spectrometric analysis ofproteins and glycans.

SUMMARY

The invention provides surfactant compounds useful for aiding thesolubilization, digestion, analysis, and/or characterization ofbiological material, for example, proteins. The compounds can also aidin the recovery of peptides generated during protein digestion. Thecompounds can improve enzymatic protein deglycosylation withoutinterfering with downstream sample preparation steps and massspectrometric analysis. The compounds are specifically useful asdigestion aids that can be decomposed by an acid, by heat, or acombination thereof. Decomposition of the surfactants allows for facileseparation from isolated samples, and/or allows for analysis of thesample without interfering with the sensitivity of various analyticaltechniques.

The surfactant compounds can also enhance the stability and reactivityof proteases, for example, trypsin and chymotrypsin, thus providing amethod for faster and more efficient cleavage of peptide bonds in asample. This increased stability and/or reactivity of the protease cantherefore reduce the amount of protease required for effective sampledigestion, and reduce the amount of time required for the digestion. Thereduction of time required for digestion thus provides a method amenableto rapid on-line, automated digestion and analysis.

Accordingly, the invention provides methods for solubilization ofproteins, including highly hydrophobic proteins (i.e., membraneproteins), protease-assisted protein digestion for proteins, forexample, for proteins ‘in solution’, and for proteins ‘in-gel’. Themethod includes combining a sample (in gel or in solution) and aprotease with a surfactant compound of the invention. The protease canbe any suitable protease for a given purpose, for example, trypsin orchymotrypsin. Other suitable proteases include serine proteases,threonine proteases, cysteine proteases, aspartic acid proteases (e. g.,plasmepsin), metalloproteases, glutamic acid proteases, or a combinationthereof.

The invention also provides methods for improved recovery of peptidesobtained in in-solution and in-gel digestion protocols. The surfactantsdescribed herein can achieve the improved recovery by preventing peptideloss due to absorption or adsorption by glassware and plastic-ware,and/or by preventing peptide loss due to precipitation, and by allowingfor improved extraction of peptides from a gel matrix for in-geldigestion.

For in-gel protein digestion, the surfactants described herein canstreamline the sample preparation protocol by combining proteindigestion and peptide extraction into a single step. The digestion andextraction can be completed within about one hour. The resultingefficiency represents a significant improvement over conventional in-gelprotocol, which typically require overnight digestion followed by 2-3hours of peptide extraction.

The surfactants described herein can also improve protein recovery inprotein extract depletion methods, such as plasma depletion methods. Inthe depletion method, abundant proteins are removed from the proteinextract or plasma. This improves analysis of low abundant proteins,detection of which is frequently compromised due to dominantinterference with highly abundant proteins. The disadvantage of typicaldepletion methods is loss of substantial amount of low abundantproteins, which are nonspecifically absorbed by highly abundantproteins. The surfactants described herein can disrupt this nonspecificabsorption. In contrast to commonly used surfactants and detergents, thesurfactants described herein lessen or eliminate impact to downstreamapplications, such as mass spectrometry, because the surfactants of theinvention can degrade during the sample preparation protocol.

Additionally, the surfactants described herein can provide a convenient‘self-degrading’ mode of action (i.e., they can degrade by hydrolysis insolution during the protocol). This ‘self-degrading’ mode of action hasnot been shown by currently known reagents used in protein samplepreparation, wherein the surfactants self-degrade by the end of thesample preparation protocol. This offers a convenient method of usingthe surfactants for the sample preparation, in which no degradation ofthe surfactant is required after sample preparation protocol iscomplete.

Accordingly, the invention provides compounds of formulae I-IX, forexample, a compound of formula I:

wherein:

Q is (C₁-C₆)alkyl, (C₆-C₁₀)aryl, (C₅-C₁₀)heteroaryl, or(C₆-C₁₀)aryl-NH(C₁-C₆)alkyl;

Y is O, S, NH, —X—C(═O)—, —C═N—, carbonyl, or —O—C(═Z)—X—;

A is aryl, aryl(C₁-C₆)alkyl, heteroaryl, or a direct bond;

Z is O or S;

X is O, NH, or S;

V is C or N;

M is H, an alkali metal, or tetra(C₁-C₂₀)alkylammonium;

L is —X—C(═Z)—X— or a direct bond;

R¹ is (C₄-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₆-C₁₆)aryl, (C₅-C₁₀)heteroaryl,(C₁-C₂₀)alkoxy(C₁-C₂₀)alkyl, (C₁-C₁₂)alkyl(C₆-C₂₀)polyalkoxy, or(C₆-C₂₀)(alkylthio)-(C₁-C₆)alkyl, or absent when V is N;

R² and R³ are each independently H or (C₁-C₂₀)alkyl; or

R² and R³ together form a 3-8 membered carbocycle ring, or a 3-8membered heterocyclic ring comprising 1, 2, or 3 N(R^(x)), S, or O; or

—V(R²)(R³)-L-R¹ is optionally —O—R¹ when A is aryl;

wherein any alkyl, alkenyl, aryl, or heteroaryl, carbocyclic ring, orheterocyclic ring, is optionally substituted with one or more (e.g., 1,2, 3, 4, or 5) (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₁₀)alkynyl,(C₃-C₁₀)cycloalkyl, (C₁-C₂₀)alkoxy, (C₁-C₂₀)alkylcarbonyl,(C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —CO₂R^(x), —SO₂R^(x), —SO₃R^(x),nitro, amino, N(R^(x))₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₁₆)aryl,(C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, or heterocyclegroups; provided that Q is not substituted with CO₂H; and

each R^(x) is independently H, (C₁-C₆)alkyl, (C₆-C₁₆)aryl, or(C₁-C₆)alkyl-(C₆-C₁₆)aryl;

or a salt thereof.

One embodiment includes methods for analyzing a sample, includingcontacting the sample with a surfactant compound of formula I. Incertain embodiments, the method includes analyzing the sample by highperformance liquid chromatography. In various embodiments, the methodincludes analyzing the sample by mass spectrometry. In some embodiments,the method includes analyzing the sample by ion-pair liquidchromatography.

Another embodiment provides a method for performing electrophoresis thatincludes contacting a sample with a surfactant compound of Formula I.The electrophoresis can be gel electrophoresis, for example,polyacrylamide gel electrophoresis, including tube, slab gel andcapillary formats of polyacrylamide gel electrophoresis. Theelectrophoresis can be free zone electrophoresis or capillaryelectrophoresis. The methods can include degrading the surfactant afterelectrophoresis. The methods can also include degrading the surfactantafter electrophoresis with an acidic solution, with heat, or acombination thereof. The methods can further include purifying thesample after degrading the surfactant.

The invention also provides a kit for performing analysis of abiomaterial sample that includes a surfactant compound of formula I. Thekit can include a component for degrading the surfactant, and optionallya molecular weight standard. The kit can also include a stainingreagent. In certain embodiments, the surfactant is incorporated into agel medium in the kit. The kit can optionally include a plastic in-geldigestion device, an enzyme, such as a protease, for example a serineprotease, such as trypsin, or chymotrypsin, or Lys-C, or a glycosidase,for example, PNGase F. The kit can also optionally include C18 clean-uptips, and reagents such as chemical tags and/or iodoacetamide.

Additionally, the invention provides a method for enhancing chemicaldigestion of a biomolecule, including contacting the molecule with adigestive enzyme and a surfactant compound of formula I, to therebyenhance the chemical digestion of the molecule.

The invention also provides methods of making the surfactant compoundsof the invention, and provides intermediates used in their preparation.

The invention also provides a method for recovery of peptides after adigestion (in-solution or, in alternatively, in-gel) is complete. Themethod allows for a dramatic increase in peptide yield resulting fromimproved peptide extraction from a gel (in in-gel digestion) and frompreventing peptide loss due to absorption by glassware or plastic-ware,or due to precipitation (in both in-solution and in-gel digestions).Peptides extracted from gels are generally limited to the size of about2,500 Da. Longer peptides largely remain trapped in the gel. Recovery ofpeptides with increased hydrophobicity is also impacted. The methodallows for increased recovery of longer and more hydrophobic peptides inamounts amenable for advanced analysis, such as MS/MS. Additionally, themethod can improve the quality of protein analysis by preservingacid-labile amino acids and acid-labile post-translationalmodifications.

The invention also provides a method to combine in-gel protein digestionand peptide extraction into a single step comprising contacting aprotein-containing gel with an aqueous solution that contains a proteaseand a compound of the invention, whereby a protein in the gelsolubilizes and unfolds to allow for digestion by the protease in theaqueous solution, and separating the aqueous solution that containsdigested peptides extracted from the gel. The presence of the compoundof the invention can improve and accelerate the digestion of the proteinin the gel, compared to known surfactants, and simultaneously enhancesthe extraction of the digested peptides from the gel and eliminates theneed for further extraction.

Additionally, the invention provides a method for enhancing enzymaticdeglycosylation of glycoproteins. Use of the surfactants describedherein can achieve the enhancing enzymatic deglycosylation ofglycoproteins without interfering with downstream sample preparationsteps and mass spectrometric analysis.

Although certain aspects, embodiments, drawings and elements of theinvention are described herein, they are meant to be illustrative andnot limiting. For example, one of ordinary skill in the art will be ableto establish equivalents to certain elements herein, and thoseequivalents are considered to be within the spirit and scope of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments or various aspectsof the invention. In some instances, embodiments of the invention can bebest understood by referring to the accompanying drawings in combinationwith the detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention, however, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1 illustrates the degradation profile of a 0.025% solution ofsurfactant 3211 at 40° C., according to one embodiment.

FIG. 2 illustrates the effect of compound 3116 on trypsin activityaccording to one embodiment, showing that compound 3116 stabilizedtrypsin activity while SDS inhibited trypsin activity within 30 minutes;trypsin activity was measured as a rate of N-α-benzoyl-L-arginine ethylester hydrochloride (BAEE) cleavage.

FIG. 3A and FIG. 3B illustrate the stimulation of protein digestionaccording to one embodiment, using compound 3116. FIG. 3A shows an HPLCchromatogram after overnight incubation of horse myoglobin with trypsinin the absence of 3116. FIG. 3B shows an HPLC chromatogram after onehour of incubation of horse myoglobin with trypsin in the presence of3116.

FIG. 4A shows a total ion chromatogram for digests of mouse membraneproteins using urea. FIG. 4B shows a total ion chromatogram for digestsof mouse membrane proteins using surfactant compound 3211.

FIG. 5 illustrates the Mascot search results for digests of mousemembrane proteins, using urea and surfactant compound 3211 separately;the count of identified proteins specific to urea and compound 3211digests, and overlapping identified proteins are illustrated.

FIG. 6 illustrates a 1 hour in-gel digestion protocol with the aid of asurfactant (compound 3211), according to one embodiment of theinvention.

FIG. 7A and FIG. 7B illustrate peptide extraction with the aid of asurfactant, according to an embodiment of the invention. FIG. 7A shows aspectrum of peptides extracted into a digestion mixture after overnightdigestion of Bovine Serum Albumin (BSA) without the aid of a surfactantof the invention. FIG. 7B shows a spectrum of peptides extracted into adigestion mixture after 1 hour of digestion with the aid of a surfactantdescribed herein, according to an embodiment of the invention.

FIG. 8A and FIG. 8B illustrate mass spectra of an in-gel digest of anapproximately 56 kD band from mouse membrane protein extract. FIG. 8Ashows the mass spectrum without the aid of surfactant 3211, where theprotein was incubated overnight, and then peptides were extractedaccording to the peptide extraction protocol. FIG. 8B shows the massspectrum with the aid of surfactant 3211, according to one embodiment,where the protein digestion and peptide extraction was complete in asingle 1 hour step. Peptides were concentrated with C18 clean-up tipsand analyzed using MALDI-TOF mass spectrometry.

FIG. 9 illustrates analysis of the protein band of FIG. 8 with LC-MS/MS.The graph shows the number of proteins identified with overnightdigestion without the aid of compound 3211. It also shows proteincoverage for each identified protein achieved with either protocol.

FIG. 10 illustrates results of 2D LC-MS/MS analysis of mouse heartmembrane proteome. The membrane protein was solubilized under threedifferent conditions: urea, surfactant or urea/surfactant mix. Eachcondition was digested with trypsin and analyzed with off-line 2DLC-MS/MS with an Agilent 1100 series LC/MSD Trap SL spectrometer.

FIG. 11A illustrates the comparative effect of compound 3116 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™ (Waters, Inc). FIG. 11Billustrates the effect of compound 3116 to aid in the solubilization anddigestion of Bacteriorhodopsin with Chymotrypsin as compared with thecommercially available surfactant RapiGest™ (Waters, Inc). In both FIG.11A and FIG. 11B, the upper chromatogram is the RapiGest™ surfactantassisted digestion reaction (control) and the lower chromatogram is theresult obtained with compound 3116. Typical reaction and HPLC conditionsare described in Example 12.

FIG. 12A illustrates the comparative effect of compound 3202 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™. FIG. 12B illustrates theeffect of compound 3202 to aid in the solubilization and digestion ofBacteriorhodopsin with Chymotrypsin as compared with the commerciallyavailable surfactant RapiGest™. In both FIG. 12A and FIG. 12B, the upperchromatogram is the RapiGest™ surfactant assisted digestion reaction(control) and the lower chromatogram is the result obtained withcompound 3202. Typical reaction and HPLC conditions are described inExample 12.

FIG. 13A illustrates the comparative effect of compound 3211 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™. FIG. 13B illustrates thecomparative effect of compound 3211 to aid in the solubilization anddigestion of Bacteriorhodopsin with Chymotrypsin as compared with thecommercially available surfactant RapiGest™, where Bacteriorhodopsin wassolubilized with RapiGest™ surfactant at 95° C. while solubilizationwith compound 3211 was accomplished at room temperature (approximately23° C.). In contrast, FIG. 13C illustrates the comparative effect ofcompound 3211 to aid in the solubilization and digestion ofBacteriorhodopsin with Chymotrypsin as compared with the commerciallyavailable surfactant RapiGest™, where solubilization was performed atroom temperature for both RapiGest™ surfactant and compound 3211. InFIG. 13A, FIG. 13B and FIG. 13C, the upper chromatogram is the RapiGest™surfactant assisted digestion reaction (control) and the lowerchromatogram is the result obtained with compound 3211. Typical reactionand HPLC conditions are described in Example 12.

FIG. 14A illustrates the comparative effect of compound 3212 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™. FIG. 14B illustrates thecomparative effect of compound 3212 to aid in the solubilization anddigestion of Bacteriorhodopsin with Chymotrypsin as compared with thecommercially available surfactant RapiGest™. In both FIG. 14A and

FIG. 14B, the upper chromatogram is the RapiGest™ surfactant assisteddigestion reaction (control) and the lower chromatogram is the resultobtained with compound 3212. Typical reaction and HPLC conditions aredescribed in Example 12.

FIG. 15A illustrates the comparative effect of compound 3224 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™. FIG. 15B illustrates thecomparative effect of compound 3224 to aid in the solubilization anddigestion of Bacteriorhodopsin with Chymotrypsin as compared with thecommercially available surfactant RapiGest™. In both FIG. 15A and FIG.15B, the upper chromatogram is the RapiGest™ surfactant assisteddigestion reaction (control) and the lower chromatogram is the resultobtained with compound 3224. Typical reaction and HPLC conditions aredescribed in Example 12.

FIG. 16A illustrates the comparative effect of compound 3228 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™. FIG. 16B illustrates thecomparative effect of compound 3228 to aid in the solubilization anddigestion of Bacteriorhodopsin with Chymotrypsin as compared with thecommercially available surfactant RapiGest™. In both FIG. 16A and FIG.16B, the upper chromatogram is the RapiGest™ surfactant assisteddigestion reaction (control) and the lower chromatogram is the resultobtained with compound 3228. Typical reaction and HPLC conditions aredescribed in Example 12.

FIG. 17A illustrates the comparative effect of compound 3266 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™. FIG. 17B illustrates thecomparative effect of compound 3266 to aid in the solubilization anddigestion of Bacteriorhodopsin with Chymotrypsin as compared with thecommercially available surfactant RapiGest™. In both FIG. 17A and FIG.17B, the upper chromatogram is the RapiGest™ surfactant assisteddigestion reaction (control) and the lower chromatogram is the resultobtained with compound 3266. Typical reaction and HPLC conditions aredescribed in Example 12.

FIG. 18A illustrates the comparative effect of compound 3271 to aid inthe digestion of Myoglobin with Trypsin as compared with thecommercially available surfactant RapiGest™. FIG. 18B illustrates thecomparative effect of compound 3271 to aid in the solubilization anddigestion of Bacteriorhodopsin with Chymotrypsin as compared with thecommercially available surfactant RapiGest™. In both FIG. 18A and FIG.18B, the upper chromatogram is the RapiGest™ surfactant assisteddigestion reaction (control) and the lower chromatogram is the resultobtained with compound 3271. Typical reaction and HPLC conditions aredescribed in Example 12.

DETAILED DESCRIPTION

Many analytical systems are sensitive to the presence of surfactants.For example, SDS and triton surfactants suppress the analyte signalduring matrix assisted laser desorption ionization mass spectrometry(MALDI-MS) analysis. Signal suppression from surfactant contamination iscontemplated to result from physical and chemical blockage of theionization/desorption process of MALDI-MS. Surfactant compositions andmethods suitable for MALDI-MS analyses, and other analyses, ofhydrophobic molecules including natural and synthetic polymers andpolypeptides/proteins, are of significant interest to researchersworking with biomaterials.

This invention relates to the treatment of a sample, such as a tissuesection, protein, or protein extract from a plant or animal, or otherorganism, with a compound or mixture of compounds that can performmulti-functional roles in the preparation of these samples for analysis,e.g., mass spectrometry or chromatography, at designed times determinedby treatment conditions. These compounds can function as a surfactant inhelping solubilize hydrophobic or other non-soluble compounds, or theycan help to unfold (denature) proteins, thereby drastically acceleratingand improving protein digestion with proteases.

The surfactant compounds disclosed herein can also function as enhancersof enzymatic deglycosylation of glycoproteins. Finally, the surfactantcompounds can aid the recovery of generated peptides by improvingpeptide extractions from a gel (in in-gel digestion) and by preventingpeptide loss due to absorption by labware, or due to precipitation. Dueto built-in cleavable bonds, appropriate treatment of the sample, forexample, with acid, base, heat, etc., can then cause decomposition ofthe agent to two or more smaller parts, each of which does notmaterially interfere with the analysis.

The resulting degraded products can often be removed from the samplemore readily than the original surfactant compound. In addition, massspectrometric sensitivity of the molecules can be significantly greaterin the presence of the surfactants than in the presence of SDS atsimilar concentrations, even in the presence of these degraded products.The invention thus has applicability in a variety of techniques thatbenefit from the initial presence and concomitant removal of asurfactant.

The invention aids in solving problems in the art associated withanalysis of proteins and peptides. Many analytical systems function bestwhen samples are aqueous or the molecules being analyzed in the sampleare solubilized in an aqueous environment. For example, bringingproteins to a soluble state is a requirement for efficient proteindigestion. Also, maintaining peptides in a soluble state is arequirement for efficient analysis by mass spectrometry or liquidchromatography.

Mass spectrometric analysis of hydrophobic molecules or molecules withsignificant hydrophobic regions (e.g., proteins or peptides) can bedifficult or problematic using currently known techniques. Thesemolecules are difficult, or sometimes essentially impossible, to suspendin aqueous solution. They tend to aggregate and precipitate out ofsolution as the hydrophobic domains interact in a manner to minimizecontact with the aqueous environment of typical MS samples preparations.The surfactants described herein can prevent these effects.

Molecules of commercial importance include polymers, hydrophilic orhydrophobic, with a particular advantage for hydrophobic polymers, suchas certain constituents of hydrophobic polypeptides, for examplemembrane associated proteins and cellular components. The typicalapproach to manipulating such molecules is to apply surfactants to bringthe hydrophobic molecule of interest out of its native environment andinto a more aqueous environment. Surfactants generally include ahydrophilic (or polar) head group and a hydrophobic tail. They mayarrange about a hydrophobic molecule with the tails interacting withhydrophobic areas on the molecule and the polar head group interactingwith water in the environment.

For example, receptor proteins are often associated with or insertedinto the plasma membrane of a cell and are generally hydrophobic innature (at least the lipid associated portions thereof). Surfactants canbe useful to isolate the receptor protein away from the plasma membrane.However, certain surfactants have also been known to interfere withMALDI-MS analysis. The addition of common surfactants, such as sodiumdodecyl sulfate, triton surfactants, and Tween, essentially eliminates amolecular signal generated by MALDI-MS, as well as electrosprayionization MS. Accordingly, the present invention provides compositionsand methods that solve these and other problems of the art.

Accordingly, the surfactants described herein offer a significantimprovement to protein sample preparation protocol. They improvesolubilization, digestion, deglycosylation, and peptide recovery. Theyachieve these effects on protein sample preparation protocol withoutinterfering with downstream sample preparation steps such as liquidchromatography and mass spec analysis. Additionally, the surfactantsdescribed herein can provide a novel, self-degrading mode of action inwhich no special manipulation of the surfactants (i.e., acid- or thermaldegradation) is required after the sample preparation protocol iscomplete.

A. Enzyme-Compatible Degradable Surfactants

The invention provides acid-labile and thermolabile surfactants thatdramatically accelerate protein digestion. The surfactants solubilizeand denature proteins intended for digestion without inhibiting trypsinactivity. The surfactants allow for rapid digestion of proteins,including the membrane protein bacteriorhodopsin. An acid- andthermolabile site in the surfactant allows for facile removal fromsolution. The surfactants can be designed, and the protocol conditionscan be optimized, in a way that the surfactants degrade within thedigestion reaction time period, thus eliminating the requirement for aseparate degradation step.

In the event that the digestion protocol is modified (for example, if auser digests proteins in shorter period than recommended) and someamount of the surfactants is still present, the surfactant can bereadily degraded with a small amount of acid or by heat, and removed bycentrifugation, or by solid phase extraction (e.g. by using an Omix® tipfrom Varian, Inc.). The acid or thermal degradation technique can renderthe surfactant innocuous to downstream liquid chromatography and massspectrometry analysis. Experiments have shown that the peptides can thenbe directly analyzed with mass spectrometry with no detectableinterference from the degraded detergent.

The detergent accelerates digestion by unfolding proteins thus providingaccess to internal protein sites for trypsin. Furthermore, experimentaldata also suggest that the detergent dramatically stabilizes trypsin. Invarious experiments, trypsin was found to retain a high level ofactivity for hours of incubation in the presence of the detergent,whereas it gradually lost activity in the absence of the detergent.

The stimulating effect of the surfactants is not only limited totrypsin. The surfactants also accelerated protein digestion withchymotrypsin. Chymotrypsin is a commercially important protease becauseit is increasingly used to digest membrane proteins. It is likely thatthe surfactants are compatible with many commercially importantproteases and can be used broadly as general protease enhancers.

In addition to improving protein digestion and stabilizing trypsin, thesurfactants have good solubilizing properties. The membrane proteinbacteriorhodopsin, which is insoluble in aqueous solutions, has beenefficiently solubilized in the presence of the surfactants. Therefore,the surfactants are useful not only for protein digestion, butpotentially for extraction of membrane and other hydrophobic proteinsfrom cells tissues.

The invention also provides a method for recovery of peptides afterdigestion (in-solution or in-gel) is complete. This surfactant assistedmethod allows for an increase in peptide yield resulting from improvedpeptide extraction from gel (in in-gel digestion) and preventing peptideloss due to absorption by glassware or plastic-ware, or due toprecipitation (for both in-solution and in-gel digestions). The methodalso simplifies peptide extraction steps for in-gel digestion bycombining digestion and extraction into a single step and betterpreserves acid-labile amino acids and acid-labile post-translationalmodifications. Additionally, the invention provides a method forenhancing enzymatic deglycosylation of glycoproteins. The invention canachieve this enhancing without interfering with downstream samplepreparation steps and/or mass spectrometric analysis.

B. Thermolabile Surfactants for Protein Analysis

While acid degradation is a simple and efficient way to removesurfactants from peptide solution, under certain conditions the acid cancause unwanted side effects such as cleavage of acid-labilepost-translational protein modifications (PTMs) or cleavage ofacid-labile peptide bonds. A supplemental approach has been developedthat retains all the advantages offered by acid-labile surfactants forprotein analysis but does not require acid to degrade the surfactantstructure. These new surfactants have excellent protein solubilizingproperties and dramatically improve protein digestion.

After digestion is complete, the surfactant can be degraded by simplyincreasing the reaction temperature. In addition, the thermolabileproperties provide a substantially unique ‘self-degrading’ mode ofaction (i.e., they can degrade by hydrolysis in solution during theprotocol). With this self-degrading mode, not shown with any currentlyused reagent for protein sample preparation, the surfactants canself-degrade by the end of the sample preparation protocol. This modeoffers a convenient method of using the surfactants for samplepreparation where no further manipulation is required for degradation ofthe surfactant after sample preparation protocol is complete.

For example, degradation of surfactant compound 3211 after proteindigestion is not required because the surfactant degrades during thedigestion process. The stability of a 1% solution of 3211 (10%degradation) is 8 hours at 23° C., 12 days at 4° C., and an estimated >3years at −20° C. However, the degradation rate of surfactant 3211increases in a typical protein digestion protocol. FIG. 1 shows thedegradation profile of a 0.025% solution of surfactant 3211 at 40° C.Less than about 5% of intact surfactant remains after only 2 hours.

When necessary, the surfactants can be degraded within a few minutes attemperatures above about 90° C., or within about 20-30 minutes at lowertemperature (e.g., about 65° C.). For example, when working with asufficiently robust sample, boiling the surfactant composition for 2-3minutes completely degrades the thermolabile surfactants. In otherinstances, the surfactant completely degrades by the end of thedigestion reaction time period, and the sample can be readily analyzed,for example, by mass spectrometry or chromatography. As degradationtypically occurs at neutral pH (in the digestion buffer), thesurfactants disclosed herein allow for better preservation ofacid-labile or alkali-labile PTMs. In addition, by eliminating the needfor the addition of acids, the degradation protocol is rendered moreuser-friendly.

While an acid is not required to degrade the peptides of interest,lowering the reaction pH with acid can accelerate the degradationprocess and allow rapid degradation at lower temperatures (e.g., about37° C.). Thus the surfactants disclosed herein offer at least twodifferent means (temperature and acid) for rendering the surfactantsinnocuous. In addition, the ability to heat degrade the surfactantoffers additional options to deactivate the protease followingdigestion, offering further alternatives to a protease digestion format.

As used herein, the following terms have the following definitions.

The language “hydrocarbon” includes substituted or unsubstituted alkyl,alkenyl, alkynyl, or aryl moieties.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl),alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkylgroups. The term alkyl further includes alkyl groups, which can furtherinclude oxygen, nitrogen, sulfur or phosphorous atoms replacing one ormore carbons of the hydrocarbon backbone. In certain embodiments, astraight chain or branched chain alkyl has 20 or fewer carbon atoms inits backbone (e.g., C₁-C₂₀ for straight chain, C₃-C₂₀ for branchedchain), and in some embodiments, six or fewer. In certain otherembodiments, the carbon chain can have 1-12 carbons, 1-10 carbons, 1-8carbons, 1-6 carbons, or 1-4 carbon atoms in its backbone. Likewise,certain cycloalkyls have from 3-8 carbon atoms in their ring structure,and in some embodiments, 5 or 6 carbons in the ring structure.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl(alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl,cycloheptenyl, cyclooctenyl), allyl or alkenyl substituted cycloalkenylgroups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. Theterm alkenyl further includes alkenyl groups which include oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkenyl group has 6 or fewer carbon atoms in its backbone(e.g., C₁-C₂₀ for straight chain, C₃-C₂₀ for branched chain). Likewise,cycloalkenyl groups may have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. In other embodiments, the carbon chain can have 1-12 carbons,1-10 carbons, 1-8 carbons, 1-6 carbons, or 1-4 carbon atoms in itsbackbone.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond. For example, the term “alkynyl”includes straight-chain alkynyl groups (e.g., ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.),branched-chain alkynyl groups, and cycloalkyl or cycloalkenylsubstituted alkynyl groups. The term alkynyl further includes alkynylgroups which include oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more carbons of the hydrocarbon backbone. In certainembodiments, a straight chain or branched chain alkynyl group has 20 orfewer carbon atoms in its backbone (e.g., C₁-C₂₀ for straight chain,C₃-C₂₀ for branched chain). In other embodiments, the carbon chain canhave 1-12 carbons, 1-10 carbons, 1-8 carbons, 1-6 carbons, or 1-4 carbonatoms in its backbone.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Alkyl groups can be lower alkyls. Unless the number of carbons isotherwise specified, “lower alkyl” as used herein means an alkyl group,as defined above, but having from one to ten carbon atoms. “Loweralkenyl” and “lower alkynyl” can have chain lengths of, for example,1-6, 2-6, 3-6, 1-4, 1-3, 2-4, 3-4, or 3 or 4 carbon atoms.

The term “acyl” includes compounds and moieties that contain the acylradical (CH₃CO—) or a carbonyl group. The term “substituted acyl”includes acyl groups where one or more of the hydrogen atoms arereplaced by for example, alkyl groups, alkynyl groups, halogens,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alknyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, and the like.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term “alkylamino” includes groups and compounds wherein the nitrogen is bound to atleast one additional alkyl group. The term “dialkyl amino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups whichinclude alkyl, alkenyl, or alkynyl groups bound to an amino group boundto a carboxy group. It includes arylaminocarboxy groups that includearyl or heteroaryl moieties bound to an amino group that is bound to thecarbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarboxy,” “alkenylaminocarboxy,” “alkynylaminocarboxy,” and“arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl andaryl moieties, respectively, are bound to a nitrogen atom which is inturn bound to the carbon of a carbonyl group.

The term “carbonyl” or “carboxy” includes compounds and moieties thatcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties that contain a carbonyl include aldehydes, ketones,carboxylic acids, amides, esters, anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietiesthat contain a carbon connected with a double bond to a sulfur atom.

The term “ester” includes compounds and moieties that contain a carbonor a heteroatom bound to an oxygen atom that is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “ether” includes compounds or moieties that contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom that is covalentlybonded to another alkyl group.

The term “thioether” includes compounds and moieties that contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom that is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomthat is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated,” e.g., perfluorinated, generally refers to amoiety, e.g., perfluorocarbons, wherein all hydrogens are replaced byhalogen atoms, e.g., fluorine.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, aminocarbonyl, alkylthiocarbonyl,alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (includingalkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Examples of heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “surfactant” refers to a surface-acting agent, or wettingagent, that lowers the surface tension of a liquid, or lowers theinterfacial tension between two liquids. A surfactant can be anamphiphilic organic compound, for example, a soap-like detergent such assodium dodecyl sulfate. Surfactants can be soluble in both organicsolvents and water. A surfactant of the invention, or a surfactant asdescribed herein, refers to a compound of any one of formulas I-IX.

The term “sample” or “biomaterial” refers to any biological material,tissue, or molecule that may be used in the methods of the invention.Examples include, without limitation, cell membranes and macromolecules,including proteins, and peptides. The sample can be a solution orextract containing a molecule or mixture of molecules that comprises atleast one biomolecule originating from a biological source that can besubjected to analysis. A sample can include a crude or a purified, e.g.,isolated or commercially obtained, sample. Further examples include, butare not limited to, inclusion bodies, biological fluids, biologicaltissues, biological matrices, embedded tissue samples, and cell culturesupernatants.

The phrase “sample-surfactant complex” refers to a complex formed by asurfactant disclosed herein and a component of the sample.

The term “electrophoresis” refers to any of the various methods ofanalyzing molecules by their rate of movement in an electric field,i.e., based on the charge to mass ratio of the molecules. Examplesinclude, but are not limited to, gel electrophoresis, polyacrylamide gelelectrophoresis, including the tube, slab gel and capillary formats ofpolyacrylamide gel electrophoresis, free zone electrophoresis andcapillary electrophoresis.

The terms “analysis” or “analyzing” refer to any of the various methodsof solubilizing, separating, detecting, isolating, purifying, and/orcharacterizing molecules, such as, e.g., intact proteins, peptides, andfragments thereof. Examples include, but are not limited to, solid phaseextraction; solid phase micro extraction; electrophoresis; massspectrometry, e.g., Matrix Assisted Laser Desorption Ionization-MassSpectrometry (MALDI-MS) or Electrospray Ionization (ESI); liquidchromatography, e.g., high performance, e.g., reverse phase, normalphase, or size exclusion chromatography; ion-pair liquid chromatography;liquid-liquid extraction, e.g., accelerated fluid extraction,supercritical fluid extraction, microwave-assisted extraction, membraneextraction, or Soxhlet extraction; precipitation; clarification;electrochemical detection; staining; elemental analysis; Edmunddegradation; nuclear magnetic resonance; infrared analysis; flowinjection analysis; capillary electrochromatography; ultravioletdetection; and combinations thereof.

The term “mass spectrometric detection” refers to any of the variousmethods of mass spectroscopy. Examples include, but are not limited to,electrospray ionization (“ESI”) and Matrix Assisted Laser DesorptionIonization (“MALDI”).

The terms “denature”, “denaturing” or “denaturation” are usedinterchangeably and include the modification of the tertiary and/orsecondary molecular structure of a biomolecule, such as a protein orDNA, by a surfactants compound described herein, heat, acid, alkali, orultraviolet radiation, so as to destroy or diminish some of the originalproperties, for example, the original three-dimensional conformation,and a specific biological activity.

The term “digestion” and the phrase “chemical digestion” refers to aprocess of breaking down a molecule, for example, a biomolecule, e.g., aprotein, into simpler chemical compounds (fragments). Chemical digestionis carried out using a digestion reagent, such as an enzyme, forexample, a protease, or by a reaction with a chemical cleavage reagent,such as cyanogen bromide (CNBr), or hydroxylamine. Proteases includeboth specific, e.g., trypsin and chymotrypsin, and nonspecific, e.g.pepsin and papain, proteases. The chemical digestion can result inbreaking of amide bonds. In certain instances, the chemical digestioncan result in breaking designated or specific amide bonds.

Surfactant Compounds of the Invention

The invention provides a compound of formula I:

wherein:

Q is (C₁-C₆)alkyl, (C₆-C₁₀)aryl, (C₅-C₁₀)heteroaryl, or(C₆-C₁₀)aryl-NH(C₁-C₆)alkyl;

Y is O, S, NH, —X—C(═O)—, —C═N—, carbonyl, or —O—C(═Z)—X—; A is aryl,aryl(C₁-C₆)alkyl, heteroaryl, or a direct bond;

Z is O or S;

X is O, NH, or S;

V is C or N;

M is H, an alkali metal, or tetra(C₁-C₂₀)alkylammonium;

L is —X—C(═Z)—X—, a direct bond, or absent when V is N;

R¹ is (C₄-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₆-C₁₆)aryl, (C₅-C₁₀)heteroaryl,(C₁-C₂₀)alkoxy(C₁-C₂₀)alkyl, (C₁-C₁₂)alkyl(C₆-C₂₀)polyalkoxy, or(C₆-C₂₀)(alkylthio)-(C₁-C₆)alkyl, or absent when V is N;

R² and R³ are each independently H or (C₁-C₂₀)alkyl; or

R² and R³ together form a 3-8 membered carbocycle ring, or a 3-8membered heterocyclic ring comprising 1, 2, or 3 N(R^(x)), S, or O; or

—V(R²)(R³)-L-R¹ is optionally —O—R¹ when A is aryl;

wherein any alkyl, alkenyl, aryl, or heteroaryl, carbocyclic ring, orheterocyclic ring, is optionally substituted with one or more (e.g., 1,2, 3, 4, or 5) (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₁₀)alkynyl,(C₃-C₁₀)cycloalkyl, (C₁-C₂₀)alkoxy, (C₁-C₂₀)alkylcarbonyl,(C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —CO₂R^(x), —SO₂R^(x), —SO₃R^(x),nitro, amino, N(R^(x))₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₁₆)aryl,(C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, or heterocyclegroups; provided that Q is not substituted with CO₂H; and

each R^(x) is independently H, (C₁-C₆)alkyl, (C₆-C₁₆)aryl, or(C₁-C₆)alkyl-(C₆-C₁₆)aryl;

or an anion or a salt thereof.

Specific values of Q include methyl, ethyl, propyl, butyl, pentyl,hexyl, and branched variations thereof. The group Q can be substituted.For example, in one embodiment, Q can be a hydroxy substituted alkyl,such as 2-propyl. In certain specific embodiments, Q can be(C₂-C₃)alkyl. In other embodiments, Q can be (C₆-C₁₀)aryl, for example,phenyl or naphthyl; or (C₅-C₁₀)heteroaryl, for example, furyl orpyridyl. In yet other embodiments, Q can be (C₆-C₁₀)aryl-NH(C₁-C₆)alkyl,for example, phenyl-amino-ethyl or phenyl-amino-propyl. In certainembodiments, the hydrogen of the (C₆-C₁₀)aryl-NH(C₁-C₆)alkyl can bereplaced with a (C₁-C₆)alkyl-SO₃-M group, such as in formula IX.

Specific values of Y include O, S, NH, —O—C(═O)—, —NH—C(═O)—, —S—C(═O)—,—C═N—, carbonyl, —O—C(═O)—O—, —O—C(═O)—NH—, —O—C(═O)—S—, —O—C(═S)—O—,—O—C(═S)—NH—, and —O—C(═S)—S—.

Specific values of A include aryl, such as phenyl or naphthyl,heteroaryl, such as furyl or pyridyl. The group A can also be a directbond linking Y to V.

A specific value for V is C. Another specific value for V is N. Thegroup A can be unsubstituted, or substituted, for example, with a haloor nitro group.

A specific value for M is H. The group M can also be an alkali metal,such as lithium, sodium, or potassium. Other values for M includetetraalkyl ammonium groups, such as tetraethyl ammonium or tetrabutylammonium. The group M can also be other ammonium groups wherein thenitrogen substituents are alkyl, aryl, or a combination thereof, such astrimethylphenyl ammonium. Other cations known to those of skill in theart may also be used as suitable counterions for the sulfate groupassociated with the group M. As would be recognized by one skilled inthe art, sodium salts of the compounds can be conveniently isolated, andthese sodium salts work well in many embodiments of the invention.

Specific values for L include —X—C(═O)—X—, and —X—C(═S)—X—, wherein X isO, NH, or S; for example, carbonate, thiocarbonate, carbamate,thiocarbamate, and derivatives thereof.

Specific values of R¹ include alkyl chains of 4-20 carbon atoms inlength, either substituted or unsubstituted. Certain specific valuesinclude pentyl, hexyl, heptyl, decyl, undecyl, dodecyl, tetradecyl, andthe like. R¹ can also be other groups including (C₂-C₂₀)alkenyl, forexample, 1-octenyl, or 1-dodecenyl; (C₆-C₁₆)aryl, for example, phenyl;(C₅-C₁₀)heteroaryl, for example, furyl, or pyridyl;(C₁-C₂₀)alkoxy(C₁-C₂₀)alkyl, for example, nonoxylmethyl;(C₁-C₁₂)alkyl(C₆-C₂₀)polyalkoxy, or (C₆-C₂₀)(alkylthio)-(C₁-C₆)alkyl,for example, nonylthiomethyl; or absent, for example, when V is N.

Values for R² include H and (C₁-C₂₀)alkyl, for example, (C₁-C₁₆)alkyl,(C₁-C₁₂)alkyl, (C₁-C₈)alkyl, (C₂-C₈)alkyl, or (C₁-C₆)alkyl. One specificvalue is H. Another specific value is methyl.

Values for R³ include H and (C₁-C₂₀)alkyl, for example, (C₁-C₁₆)alkyl,(C₁-C₁₂)alkyl, (C₁-C₈)alkyl, (C₂-C₈)alkyl, or (C₁-C₆)alkyl. One specificvalue is H. Another specific value is methyl.

The groups R² and R³ can also be combined together to form a 3-8membered ring. The ring can be a carbocycle ring, or a heterocyclicring. The heterocyclic ring can include one or more, e.g., 1, 2, or 3heteroatoms, including N, S, O, or a combination thereof. A nitrogen ofthe heterocyclic ring can be substituted with an H, or an substituent,for example, a (C₁-C₆)alkyl, a (C₆-C₁₆)aryl, or a(C₁-C₆)alkyl(C₆-C₁₆)aryl group. Examples of heterocyclic rings include,but are not limited to, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine.

In certain embodiments, the group —V(R²)(R³)-L-R¹ can be —O—R¹ when A isan aryl group. For example, in certain embodiments, —V(R²)(R³)-L-R¹ canbe —O—R¹ wherein R¹ is (C₄-C₂₀)alkyl or (C₁-C₂₀)alkoxy(C₁-C₂₀)alkyl. Inone embodiment, Q can be (C₁-C₆)alkyl, Y can be —C═N—, A can be phenyl,and —O—R¹ can be pentoxy, hexyloxy, decyloxy, or tetradecyloxy.

In various embodiments, the alkyl, alkenyl, aryl, or heteroaryl,carbocyclic ring, or heterocyclic ring, can be unsubstituted, oroptionally substituted with one or more (e.g., 1, 2, 3, 4, or 5)substituents as defined herein. Various substituents include(C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₁₀)alkynyl, (C₃-C₁₀)cycloalkyl,(C₁-C₂₀)alkoxy, (C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo,hydroxyl, —CO₂R^(x), —SO₂R^(x), —SO₃R^(x), nitro, amino, N(R^(x))₂,mercapto, (C₁-C₂₀)alkylthio, (C₆-C₁₆)aryl, (C₆-C₃₀)arylthio,trifluoromethyl, ═O, heteroaryl, or heterocycle groups.

Each variable R^(x) can independently be H, (C₁-C₆)alkyl, (C₆-C₁₆)aryl,or (C₁-C₆)alkyl-(C₆-C₁₆)aryl. For example, in one embodiment, R^(x) canbe H. In another embodiment, R^(x) can be methyl, ethyl, propyl, butyl,pentyl, or hexyl, wherein each is optionally branched, optionallyunsaturated, and/or optionally substituted with one of the groupsdescribed above. In another embodiment, R^(x) can be phenyl, optionallysubstituted with one of the groups described above. In yet anotherembodiment, R^(x) can be benzyl, phenylethyl, phenylpropyl, or the like,optionally substituted with one of the groups described above.

In certain embodiments, Q is not substituted with one of theaforementioned substituents. For example, in one embodiment, Q is notsubstituted with CO₂H.

In certain embodiments, a salt of an acid can be prepared. Suitablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The salts includeconventional salts and quaternary ammonium salts of the parent compoundformed, for example, from inorganic or organic acids. Salts can includethose derived from inorganic acids such as hydrochloric, hydrobromic,hydroiodic, sulfuric, sulfamic, phosphoric, nitric and the like. Saltsprepared from organic acids can include those such as acetic,2-acetoxybenzoic, ascorbic, benzenesulfonic, benzoic, citric,ethanesulfonic, ethane disulfonic, formic, fumaric, gentisinic,glucaronic, gluconic, glutamic, glycolic, hydroxymaleic, isethionic,isonicotinic, lactic, maleic, malic, methanesulfonic, oxalic, pamoic(1,1′-methylene-bis-(2-hydroxy-3-naphthoate)), pantothenic,phenylacetic, propionic, salicylic, sulfanilic, toluenesulfonic,stearic, succinic, tartaric, bitartaric, and the like. Certain compoundscan also form salts with various amino acids.

The compounds of the invention can also be certain compounds of formulasII-IX. For example, certain compounds formula I can also be certaincompounds of formulas II-VIII. For certain specific compounds of theinvention, i.e., compounds of formulas I-IX, see the compoundsillustrated in the Examples section below.

In one embodiment, the compound of formula I is a compound of formulaII:

wherein the variables are as described above for formula I. In oneembodiment, R¹ is heptyl or undecyl, R² and R³ are both methyl, Z is O,X is NH, Q is (C₁-C₆)alkyl, for example, propyl, and M is H. In someembodiments, R¹ is substituted with any aryl or heteroaryl group. Thearyl or heteroaryl group can be substituted. In certain embodiments, thearyl or heteroaryl substituent is located alpha (one carbon away) fromthe carbon to which R² and R³ are attached. In one specific embodiment,R² and R³ are both H and R¹ is methyl, substituted with phenyl, whereinthe phenyl is substituted with hexyloxy.

In another embodiment, the compound of formula I is a compound offormula III:

wherein the variables are as described above for formula I.

In another embodiment, the compound of formula I is a compound offormula IV:

wherein the variables are as described above for formula I, and R⁴ andR⁵ are each independently H or (C₁-C₂₀)alkyl.

In another embodiment the compound of formula I is a compound of formulaV:

wherein the variables are as described above for formula I.

In another embodiment, the compound of formula I is a compound offormula VI:

wherein the variables are as described above for formula I.

In another embodiment, the compound of formula I is a compound offormula VII:

wherein the variables are as described above for formula I, and thecarbon attached to R² and R³ can be in an ortho, meta, or paraorientation with respect to the oxy substituent of the benzene ringshown in formula VII.

In another embodiment, the compound of formula I is a compound offormula VIII:

wherein the variables are as described above for formula I, and theR¹—O— substituent of the benzene ring in formula VIII can be in anortho, meta, or para orientation with respect to the imine substituentof the benzene ring.

In another embodiment, the surfactant compound is a compound of formulaIX:

wherein:

Q is (C₁-C₆)alkyl, (C₆-C₁₀)aryl, or (C₅-C₁₀)heteroaryl;

Y is O, S, NH, —X—C(═O)—, —C═N—, carbonyl, —O—C(═Z)—X—, or —O—(CH₂)₁₋₆—;

A is aryl, aryl(C₁-C₆)alkyl, heteroaryl, or a direct bond;

Z is O or S;

X is O, NH, or S;

V is C or N;

each M is independently H, an alkali metal, ortetra(C₁-C₂₀)alkylammonium;

L is —X—C(═Z)—X— or a direct bond;

R¹ is (C₄-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₆-C₁₆)aryl, (C₅-C₁₀)heteroaryl,(C₁-C₂₀)alkoxy(C₁-C₂₀)alkyl, (C₁-C₁₂)alkyl(C₆-C₂₀)polyalkoxy, or(C₆-C₂₀)(alkylthio)-(C₁-C₆)alkyl, or absent when V is N, or optionally Hwhen R² and R³ together form a ring;

R² and R³ are each independently H or (C₁-C₂₀)alkyl; or

R² and R³ together form a 3-8 membered carbocycle ring, or a 3-8membered heterocyclic ring comprising 1, 2, or 3 N(R^(x)), S, or O; or

—V(R²)(R³)-L-R¹ is optionally when A is aryl, or —C(aryl)₂(aryl)-OR¹;

R⁴ is —(C₁-C₁₂)alkyl-SO₃-M;

R⁵ is H or —(C₁-C₁₂)alkyl-SO₃-M;

wherein any alkyl, alkenyl, aryl, or heteroaryl, carbocyclic ring, orheterocyclic ring, is optionally substituted with one or more (e.g., 1,2, 3, 4, or 5) (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₁₀)alkynyl,(C₃-C₁₀)cycloalkyl, (C₁-C₂₀)alkoxy, (C₁-C₂₀)alkylcarbonyl,(C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —CO₂R^(x), —SO₂R^(x), —SO₃R^(x),nitro, amino, N(R^(x))₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₁₆)aryl,(C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, or heterocyclegroups; provided that Q is not substituted with CO₂H; and

each R^(x) is independently H, (C₁-C₆)alkyl, (C₆-C₁₆)aryl, or(C₁-C₆)alkyl-(C₆-C₁₆)aryl;

or a salt thereof.

In one specific embodiment, an acid-labile surfactant of formula Iincludes the compound 3116:

Compound 3116 can be used as an acid-labile surfactant to facilitate thesolubilization and denaturation of a protein or peptide. Compound 3116stabilizes proteases and enhances the proteolysis activity of trypsin.See FIG. 2, which illustrates the effect of compound 3116 on trypsinactivity. Under standard digestion conditions, compound 3116 stabilizedtrypsin activity while SDS inhibited trypsin activity within 30 minutes.Additionally, after degradation, compound 3116 does not interfere withchromatographic separations and mass spectrometric analyses.

For example, digestion of myoglobin using standard protocol requiresovernight digestion. In one experiment, myoglobin was largely digestedwithin one hour in the presence of compound 3116. Horse myoglobin wasdigested with trypsin at a 50:1 ratio for one hour at 37° C., alone andseparately in the presence of 0.01% compound 3116.

FIG. 3A and FIG. 3B shows HPLC chromatograms of the myoglobin digests:FIG. 3A, standard protocol with no 3116, and FIG. 3B, digest in thepresence of 3116. The peaks in FIG. 3A and FIG. 3B indicate peptides orpartial digestion fragments. As can be observed from the chromatograms,the standard protocol resulted in insufficient digestion while thedigestion in the presence of compound 3116 produced numerous (>15)identifiable peaks.

In another specific embodiment, an acid-labile and thermolabilesurfactant of formula I includes compounds 3211 and 3212:

Compounds 3211 and 3212 can be used as thermolabile and/or acid-labilesurfactants to facilitate the solubilization and denaturation of apeptide. For any of the compounds described herein, the sulfonic acidform of the compound may be employed, or alternatively, a sulfonic acidsalt may be employed, depending on the desired properties and theconditions employed. For example, in certain buffer solutions thecompounds disclosed herein will convert to, or be in an equilibriumwith, an alkali metal salt, such as a sodium salt.

In another specific embodiment, an acid-labile surfactant of formula Iincludes the compound 3266:

Compound 3266 is an acid-labile surfactant that can be used as an aidfor deglycosylation of a biomaterial in conjunction with a glycosidase.

The surfactants described herein offer substantial improvement inprotein sample preparation for mass spectrometric analysis. Thesesurfactants therefore provide significant advantages for those involvedin the use of digestion enzymes for the analysis and identification ofpeptide samples, including the ability to degrade the surfactant suchthat the degraded surfactant components do not interfere in theanalytical procedure.

Methods of Using the Surfactants

Cellular protein extracts are typically too complex for immediate massspectrometric analysis. Two major approaches to reducing proteincomplexity include fractionation in gel, and fractionation with liquidchromatography. The surfactant compounds disclosed herein can aid inboth methods of reducing protein complexity, both prior to and aftersample digestion. The surfactant compounds aid the solubilization anddigestion of proteins, and can be used as a replacement for other agentsthat interfere with fractionation and chromatography, and otheranalytical techniques.

The invention provides methods of protein sample preparation andanalysis. The method can include sample fractionation followed by gelelectrophoresis, for example, using 1D or 2D SDS-PAGE. The separatedproteins can be excised and then individually digested, for example, bychemical digestion agents or a protease, such as trypsin, in conjunctionwith a surfactant compound as described herein, to provide a mixture ofpeptide samples. These samples can then be analyzed and characterized byvarious techniques, for example, HPLC or mass spectrometry, such asMALDI-TOF mass spectrometry. The surfactants described herein bring animportant advantage to existing in-gel digestion protocols. Acting asprotein digestion aid and, simultaneously, as a peptide extractingagent, the surfactants allow for the combination of in-gel proteindigestion and peptide extraction into a single and short step.

The methods of the invention also include protein digestion aided by asurfactant of the invention, followed by chromatographic separation. Theseparated peaks can then be analyzed by mass spectrometry, for exampleESI. The LC/MS techniques can be conducted in a high through-put format.The mass to charge (m/z) ratios of the peptides can be recorded andselected for MS/MS analysis. The peptides can then be dissociated intofragments representative of the original peptide sequence.

In one aspect, the invention provides methods for analyzing a sample bycontacting a sample with a surfactant compound of any one of formulasI-IX as defined above, and analyzing the sample. In certain embodiments,the sample may be heated either before or after contacting the samplewith a surfactant of the invention.

In various embodiments, analyzing a sample includes electrophoresis. Incertain embodiments, the electrophoresis is gel electrophoresis, freezone electrophoresis, or capillary electrophoresis. The electrophoresiscan be polyacrylamide gel electrophoresis, including tube, slab gel, andcapillary formats of polyacrylamide gel electrophoresis.

In other embodiments, the step of analyzing the sample includes massspectrometric analysis, high performance liquid chromatography, ion-pairliquid chromatography, liquid-liquid extraction, ultraviolet detection,or a combination thereof.

In various embodiments, hydrolysable surfactants can be used to complexwith protein mixtures for polyacrylamide gel electrophoresis. After theelectrophoretic separation, the proteins can be freed from surfactants,optionally by treating the gel with acid solution, or by heating thegel. The protein mixtures may be further purified by conventionalseparation methods such as liquid-liquid extraction, solid-phaseextraction or liquid chromatography.

The invention also provides a kit for performing analysis of a sample,where the kit includes a surfactant compound as described herein,instructions for use, and optionally a solution for degrading thesurfactant, a gel, a molecular weight standard, and/or a stainingreagent.

The invention provides a simplified, yet more efficient method ofextracting gel-trapped peptides. The surfactants of the inventiongreatly improve the overall yield of peptides in an in-gel digestionprotocol. They also offer a important advantage over standard peptideextraction protocols, wherein the surfactants allow for extraction oflong and/or very hydrophobic peptides that regularly remain in a gelafter standard peptide extraction (i.e., with TFA and/or acetonitrile).The method can also increase peptide yield by other mechanisms, such asby preventing peptide loss due to absorption of peptides by labware(i.e., plastic tips and reaction tubes, glass pipettes, and the like) orby precipitation of the peptides. Both in-gel and in-solution digestionprotocols benefit from these advantageous properties of the methodsdescribed herein.

In certain embodiments, a chemical reaction using surfactants of theinvention is chemical digestion. In one embodiment, the chemicaldigestion occurs by contacting a molecule, e.g., a biomolecule, with aprotease. Exemplary proteases include, but are not limited to specificproteases such as Trypsin, Chymotrypsin Lys-C, Glu-C (V8 protease),AspN, Arg-C, S. Aureus, and Clostripain, and non-specific proteases,such as, Pepsin, and Papain. In certain embodiments the protease isimmobilized, e.g., immobilized enzymatic reactor. Alternatively,digestion may be accomplished by reaction with CNBr or reaction withhydroxylamine. Additionally, in certain embodiments, the digestion canoccur in an electrophoretic gel in the presence or absence of one ormore surfactants that are different from the surfactants of theinvention, e.g., SDS.

Solubilization of insoluble samples by surfactants of the presentinvention allows digestion of samples that are typically insoluble andtherefore difficult to digest by known methodology. In addition, thesurfactants enable the use of lower amounts of trypsin to digest aprotein. For example, the ratio of trypsin to total protein is typically1:50 to 1:20. However, in some embodiments, in the presence of thesurfactants described herein, the ratio of trypsin to total protein canbe 1:100 or lower. Furthermore, use of the surfactants described hereincan increase the digestion rate of trypsin and result in fewerincomplete cleavages than observed for known methodologies, such as theaddition of organic solvents or use of excessive heat.

Additionally, it is known that if digestion, in which urea has beenutilized as a solubilizing agent, is allowed to proceed for too long,the urea will act upon and modify the protein, making analysis ofpeptide fragments more difficult. In this regard, the surfactants of theinvention will not modify the protein, regardless of the length of timeallowed for digestion.

The invention offers an advantageous improvement to standard in-solutiondigestion protocol involving urea as a solubilizing agent. A user hasthe option to mix urea and a surfactant as described herein, and digestproteins in such a mixture. The surfactants retain their usefulproperties (solubilization, denaturing activity, and peptidepreservation) in the presence of urea. Protein digestion in aurea/surfactant mixture can lead to a higher number of identifiedproteins, higher number of generated peptides per protein, and higherpeptide recovery, than in the presence of urea or surfactant alone. Oneparticularly attractive feature of the methods described herein is thatthere is no need to alter the digestion protocol. The surfactants arefully compatible with standard in-solution digestion protocols.

In certain embodiments, use of the surfactants described herein canenhance a chemical reaction or enhance a chemical property of anotheragent. Such chemical reactions and properties include a more completereaction or digestion, increased digestion efficiency, increaseddigestion yield, increased digestion rate, and increased utility, suchas reduced interference in spectrometric or chromatographic analysistechniques.

After a chemical reaction, a sample can be separated from thesurfactants by various methods, including degradation with acidicsolution. The sample may be further purified by conventional separationmethods such as liquid-liquid extraction, solid-phase extraction orliquid chromatography. This ability to separate a sample fromsurfactants easily after a chemical reaction may be used in variousapplications, with significant benefits to separation science.

An additional embodiment provides a method for enhancing chemicaldigestion of a biomolecule comprising contacting the molecule with adigestive enzyme, e.g., a protease, and a surfactant of the presentinvention, to thereby enhance the chemical digestion of the molecule. Inan additional embodiment, the invention provides a kit for enhancingchemical digestion of a biomolecule comprising a surfactant of thepresent invention, and instructions for use. In certain embodiments, thebiomolecule is a protein.

In some embodiments, the kit for enhancing chemical digestion of abiomolecule further includes a digestive enzyme, e.g., a protease and/ora glycosidase. Suitable proteases include, without limitation, Trypsin,Chymotrypsin Lys-C, Glu-C(V8 protease), AspN, Arg-C, S. Aureus,Clostripain, Pepsin, and Papain. A suitable glycosidase can be PNGase F.

In various embodiments, the products of surfactant degradation arecompatible with mass spectrometric detection, high performance liquidchromatography analysis, and with protease activity. In certainembodiments, a protein fragment is generated by chemical digestion or acombination of chemical alteration and chemical digestion. In certainspecific embodiments, a protein fragment or a peptide is the product ofa protein that has been digested by contact with a protease and asurfactant as described herein.

In an additional embodiment, compounds of the invention may be used inone dimensional and two dimensional polyacrylamide gel electrophoresis.Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) is atechnique commonly used for the analysis of mixtures of proteins. (U. KLaemmli, Nature 227, 680-685, 1970). Proteins are separated first by anelectrophoretic such as isoelectric focusing followed by a seconddimension separation based on protein size.

Sodium dodecyl sulfate (SDS), the detergent most often used with2D-PAGE, forms stable non-covalent complexes with proteins. The SDScomplexed proteins have identical charge density, therefore, theyseparate in an electrical field according to their size. This techniqueis capable of separating a complex protein mixture into several hundredindividual components that can be excised from the gel and furtheridentified by other techniques. One such technique is mass spectrometry.

The direct analysis of proteins removed from electrophoresis gels isoften difficult. Commonly, the samples contain detergent concentrationsthat hinder analysis by mass spectrometry. In MALDI analysis forexample, this problem is the result of the tendency of the detergent toaggregate or associate with the protein or peptide preventing properincorporation into the matrix crystal. Special steps must be taken toremove the interference prior to analysis by MALDI-MS. Examples of suchmeasures include, but are not limited to electroblotting of PAGE gelsand detergent exchange of SDS with a more MALDI tolerant detergent liken-octyl-glucoside, for example. An alternative approach is to useacid-labile surfactants disclosed herein in place of commonly useddetergents in SDS-PAGE, for example, the surfactants disclosed in theExamples below.

Furthermore, the ability to estimate the size and amount of a proteinhas led to various applications of SDS-PAGE. However, there are somedrawbacks to the technology. For example, it is very difficult to usemass spectrometry to monitor and analyze samples from SDS-PAGEseparations because SDS interferes with the sensitivity of massspectrometry detection. Furthermore, it is very difficult to separateSDS from SDS/protein complex since SDS is a surfactant that formsemulsions. The compounds of the instant invention provide a solution tothe problems associated with using SDS prior to mass spectrometryanalysis.

The surfactants of the invention may be used in applications whichbenefit from the initial presence and ultimate removal of a surfactant.In particular, the surfactants described herein are useful for thesolubilization, degradation, digestion, separation, purification,analysis, and/or characterization of peptides and proteins.

The surfactants of the invention are particularly advantageous forin-gel protein digestion. The in-gel digestion method based on use ofthe surfactants is fully compatible with SDS-based gel electrophoresis.After resolving proteins in SDS-PAGE, a gel slice with a protein ofinterest is cut out of the gel and SDS is removed by standard washingtechniques. The gel slice is soaked in a solution containing thesurfactant, or the surfactant and trypsin, or other protease. Thesurfactants can then act in three ways. First, they can solubilizeproteins precipitated within a gel during the fixation step. Second,they can unfold the proteins, allowing for easy access to proteincleavage sites for proteases. Third, they can allow for efficientextraction of generated peptides from the gel.

One particular advantage is that the surfactants allow for extraction oflong and/or very hydrophobic peptides that typically remain in a gelthat is treated with a standard peptide extraction protocol (i.e., withTFA and acetonitrile). Additionally, use of the surfactants ensures fullpeptide recovery after extraction by preventing peptide absorption bylabware (i.e., pipette tips and reaction tubes), or by preventingpeptide precipitation due to poor solubility.

The surfactants of the invention can provide an important advantage toexisting in-gel digestion protocol. Acting as protein digestion aidsand, simultaneously, as peptide extracting agents, the surfactants allowone to combine in-gel protein digestion and peptide extraction into asingle efficient step. The step can be completed in one hour.

These advantages make the surfactants ideal universal agents for aidingin-gel sample preparation. Their use results in a reduction in the timeand labor required for the sample preparation, as well as an increase insequence coverage and probability scores. In several embodiments, noseparate degradation step is required for the surfactants because theyself-degrade during a digestion reaction or during downstream samplepreparation steps (i.e., during decreasing of pH by TFA before clean-upon C18 clean-up tips). The degraded surfactants do not interfere withliquid chromatography and mass spectrometric analysis, furtherestablishing the surfactants' compatibility with comprehensive proteinanalytical techniques.

The surfactants are also suitable with in-solution digestion protocols.First, the surfactants efficiently solubilize proteins. Solubilizationis required for efficient protein digestion. Second, the surfactantsunfold proteins, allowing for easy access to protein cleavage sites forthe proteases. Third, they stabilize the protease, leading to anadditional increase in protein digestion efficiency over standardtechniques. Additionally, and potentially of most significance, thesurfactants ensure robust recovery of peptides generated in thedigestion step. The surfactants achieve this result by preventingpeptide absorption by labware. Peptide loss is a commonly observedeffect found in in-solution digestion protocol s and potentially leadsto a decrease in the number of identified proteins, individual proteinsequence coverage, and data from mass spectrometric analysis.

Additional advantages of the surfactants described herein include thatprecipitation of peptides can be avoided by adjusting the surfactantconcentration, thereby reducing or eliminating losses of peptides duringrecovery steps. Currently used acid-labile surfactants such as RapiGest′have several disadvantages, such as the formation of cloudy solutions orsuspensions, which result in peptide loss through precipitation. Also,some currently used acid-labile surfactants require boiling for completeprotein solubilization, especially for membrane proteins. Thesurfactants described herein can solubilize even the problematicmembrane proteins. Additionally, currently used surfactants provide lessenhancement of enzyme activity under mild conditions (i.e., at roomtemperature). Other currently used acid-labile surfactants demonstratelow-efficient enhancement of protein digestion. For example, underconditions where surfactant 3211 afforded complete digestion ofmyoglobin in one hour, PPS Silent′ Surfactant (obtainable from ProteinDiscovery, Knoxville, Tenn.) provided only 5% digestion. Finally,currently used commercial reagents require an additional degradationstep after protease digestion, while the surfactants described hereincan degrade under the digest conditions by the end of the digestion,greatly simplifying sample preparation.

General Preparatory Methods

Several methods exist for the preparation of urethanes (or carbamates).The method used to prepare the compounds described herein involves theconversion of an alcohol to a reactive p-nitrophenyl carbonate, followedby the addition of an amine, to provide the urethane. One skilled in theart will readily recognize that there are other ways in which thecompounds may be prepared. For example, an alcohol could be treated with1,1′-carbonyldiimidazole to provide an imidazolide, followed by additionof an amine. The amine can be, for example, a sulfonic acid sodium saltlinked to the amine through an organic group. An alcohol can also betreated with phosgene or a phosgene equivalent (e.g., diphosgene ortriphosgene) to provide a chloroformate, followed by addition of anamine. Alternatively, an alcohol can be combined with a carbamoylchloride to afford the urethane. It should be understood that manyvariations and modifications may be made while remaining within thescope of the invention.

One method for the preparation of compounds of the invention can berepresented by the procedure of Scheme 1.

wherein Gp of compound A can be any group that can be theoxy-substituent of an ester, such as an alkyl, aryl, heteroaryl, or thelike; R is any organic group that forms a suitable Grignard reagent, forexample an R² group as defined for formula I; and R¹ and Q are asdefined for formula I. Many aspects of Scheme 1 can be varied to preparethe compounds of formulas I-IX, depending on the desired product, thereactivity of the intermediates, and the necessity or convenience ofprotecting groups. Many other approaches to the preparation ofcarbamates can be taken, as discussed above.

The methods of synthesis of the invention can produce isomers in certaininstances. Although the methods of using surfactants of the invention donot always require separation of these isomers, such separation may beaccomplished, if desired, by methods known in the art. For example,preparative high performance liquid chromatography methods may be usedfor isomer purification, for example, by using a column with a chiralpacking.

Additional background information may be found in the followingpublications: Kyte et al., J. Mol. Biol. (1982) 157(1):105-32; March'sAdvanced Organic Chemistry Reactions, Mechanisms, and Structure,5.sup.th Ed. by Michael B. Smith and Jerry March, John Wiley & Sons,Publishers; Wuts et al. (1999) Protective Groups in Organic Synthesis,3.sup.rd Ed., John Wiley & Sons, Publishers; Behforouz, M.; Kerwood, J.E. Alkyl and Aryl Sulfenimides. J. Org. Chem., 34 (1), 51-55 (1969); andHarpp, D. N.; Ash, D. K.; Back, T. G.; Gleason, J. G.; Orwig, B. A.;VanHorn, W. F. A New Synthesis of Unsymmetrical Disulifdes. TetrahedronLetters, 41, 3551-3554 (1970).

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the present invention could be practiced. It should be understoodthat many variations and modifications may be made while remainingwithin the scope of the invention.

EXAMPLES Example 1. Preparation of Compound 3116

Methyl laurate (2.0 g, 9.33 mmol) was dissolved in 20 mL of anhydrousTHF and the solution was cooled in an ice-water bath. A solution of 3Mmethylmagnesium chloride in THF (6.5 mL, 19.6 mmol) was added dropwisevia syringe and the stirred reaction mixture was allowed to warm toambient temperature and react for 4 hours. The reaction mixture waspoured into 50 mL of 2M aqueous sulfuric acid solution and was extractedwith ethyl acetate. The organic extracts were dried over anhydroussodium sulfate and concentrated to give 2.0 g (99% yield) of2-methyl-2-tridecanol as a colorless oil.

MS (ESI+): m/z 215.4 (MH+).

The 2-methyl-2-tridecanol (0.36 g, 1.68 mmol) was dissolved in 1 mL ofpyridine and 2 mL of THF. The solution was cooled in an ice-water bathand p-nitrophenyl chloroformate (340 mg, 1.68 mmol) was added and thereaction was allowed to warm to ambient temperature and react overnight.The reaction mixture was roto-evaporated and the residue was partitionedbetween dichloromethane and water. The aqueous phase was extracted twicewith dichloromethane and the combined organic extracts were then driedover anhydrous sodium sulfate and roto-evaporated to provide a thickoil. The crude product was chromatographed on silica gel usingdichloromethane to afford 0.13 g (20% yield) of 2-methyl-2-tridecanolp-nitrophenyl carbonate.

To a solution of 2-methyl-2-tridecanol p-nitrophenyl carbonate (0.11 g,0.29 mmol) in 5 mL of THF was added a solution of 3-aminopropanesulfonic acid, sodium salt (0.15 g, 0.93 mmol) in 2 mL of water. Thereaction mixture was heated at 50° C. for 12 hours. The reaction mixturewas concentrated to a crude solid residue which was purified bychromatography on silica gel using a 4:1 mixture ofdichloromethane-methanol to afford 70 mg (60% yield) of compound 3116 asan off-white solid.

MS (ESI⁻): m/z 378.6 (M-H)⁻.

A synthetic route to compound 3116 is shown in Scheme 2.

Example 2. Preparation of Compounds 3211 and 3212

2-Furaldehyde (13.4 g, 0.14 mol) was dissolved in 500 mL dry THF and thesolution was cooled to 0° C. Decyl magnesium bromide (150 mL of a 1.0 Msolution in diethyl ether, 0.15 mol) was added dropwise to the cooledsolution, and the reaction allowed to stir overnight. TLC analysis (4:1ethyl acetate-heptane) indicated the reaction was complete. The reactionmixture was quenched with butanol, followed by water and the mixture wasfiltered through a sintered-glass funnel and concentrated. The crudeproduct was purified by chromatography on silica gel using a mixture of4:1 heptane-ethyl acetate to provide 29.7 g (89% yield) of1-(furan-2-yl)undec-1-ol. 1-(Furan-2-yl)undec-1-ol (4.0 g, 16.8 mmol)was dissolved in 150 mL dry THF and cooled to 0° C. p-Nitrophenylchloroformate (6.76 g, 33.5 mmol) was added and stirred to dissolve.Pyridine (15 mL) was added dropwise over 20 minutes and the reaction wasallowed to stir 1 hour, then analyzed by TLC (7:3 heptane-ethylacetate). The reaction mixture was filtered to remove pyridine HClsalts. The reaction mixture was evaporated to an oil, co-evaporatedtwice with acetonitrile, triturated with 100 mL of heptane, and filteredto remove precipitate. The heptane filtrate was evaporated to provide acrude oil. 3-Amino propane sulfonic acid, tetrabutylammonium salt wasdissolved in 100 mL of THF and added to the crude oil. The reaction wasallowed to stir 1 hour and then analyzed by TLC (9:1dichloromethane-methanol). The reaction mixture was evaporated to acrude oil and dissolved in 80 mL of water, then filtered to removeprecipitate. The resulting solution was passed through 160 grams ofcation exchange resin (Diaion® UBK 550, Mitsubishi Chemical Corporation)to convert the product to the sodium salt. The appropriate fractionswere combined and lyophilized on a freeze dryer. The fluffy yellow solidwas then chromatographed on silica gel using dichloromethane, then 85:15dichloromethane-methanol to afford a 5.6 g (78% yield) of compound 3211as a white solid.

p-Anisaldehyde (6.12 g, 45.0 mmol) was dissolved in 250 mL dry THF andthe solution was cooled to 0° C. Decyl magnesium bromide (50 mL of a 1.0M solution in diethyl ether, 50 mmol) was added dropwise to the cooledsolution, and the reaction allowed to stir overnight. TLC analysis (4:1ethyl acetate-heptane) indicated the reaction was complete. The reactionmixture was quenched with butanol, followed by water and the mixture wasfiltered through a sintered-glass funnel and then evaporated. The crudeproduct was purified by chromatography on silica gel using a mixture of4:1 heptane-ethyl acetate to provide 10.8 g (86% yield) of1-(4-methoxyphenyl)undecan-1-ol. 1-(4-Methoxyphenyl)undecan-1-ol (2.0 g,7.2 mmol) was dissolved in 40 mL dry THF and cooled to 0° C.p-Nitrophenyl chloroformate (2.17 g, 10.8 mmol) was added and stirred todissolve. Pyridine (5 mL) was added dropwise over 20 minutes and thereaction was allowed to stir 2 hours, then analyzed by TLC (7:3heptane-ethyl acetate). The reaction mixture was filtered to removepyridine HCl salts. The reaction mixture was evaporated to a crude oilthat was chromatographed on silica gel using 4:1 heptane-ethyl acetate.The resulting 1-(4-methoxyphenyl)undecan-1-ol p-nitrophenyl carbonatewas dissolved in 50 mL of THF and treated with 3-aminopropanesulfonicacid, sodium salt (1.35 g, 8.38 mmol). A few drops of water were addedto dissolve the reactants. The reaction was allowed to stir overnight,and then analyzed by TLC (9:1 dichloromethane-methanol). The reactionmixture was evaporated to a crude oil that was then chromatographed onsilica gel using dichloromethane, then 4:1 dichloromethane-methanol.Appropriate fractions were combined, filtered and evaporated to afford2.0 g (60% yield) of compound 3212 as a white solid.

Example 3. Preparation of Compound 3266

3,4-Dimethoxybenzaldehyde (3.15 g, 19.0 mmol) was dissolved in 100 mLdry THF and the solution was cooled to 0° C. Dodecyl magnesium bromide(20 mL of a 1.0 M solution in diethyl ether, 20 mmol) was added dropwiseto the cooled solution, and the reaction allowed to stir overnight. TLCanalysis (4:1 ethyl acetate-heptane) indicated the reaction wascomplete. The reaction mixture was quenched with water and the mixturewas filtered through a bed of Celite (diatomaceous earth) withheptane-ethyl acetate washes and then evaporated. The crude product waspurified by chromatography on silica gel using a mixture of 3:1heptane-ethyl acetate to provide 5.1 g (80% yield) of1-(3,4-dimethoxyphenyl)undecan-1-ol.

1-(3,4-Dimethoxyphenyl)undecan-1-ol (2.3 g, 6.8 mmol) was dissolved in20 mL dry THF and to the solution was added p-Nitrophenyl chloroformate(1.64 g, 8.2 mmol), followed by pyridine (2.0 mL). The reaction wasallowed to stir 3 hours, then analyzed by TLC (4:1 heptane-ethylacetate). The reaction mixture was filtered through Celite withheptane-ethyl acetate washings. The filtrate was evaporated to a crudeoil that was chromatographed on silica gel using 85:15 heptane-ethylacetate. The resulting 1-(3,4-dimethoxyphenyl)undecan-1-ol p-nitrophenylcarbonate 1.54 g, 3.1 mmol) was dissolved in 15 mL of THF and treatedwith a solution of 3-aminopropanesulfonic acid, sodium salt (0.64 g, 4.0mmol) in 2 mL of water. The reaction was allowed to stir overnight, andthen analyzed by TLC (4:1 dichloromethane-methanol). The reactionmixture was evaporated to a crude oil that was then chromatographed onsilica gel using 85:15 dichloromethane-methanol. Appropriate fractionswere combined and evaporated to afford 0.48 g (30% yield) of compound3266 as a white solid.

Example 4. Protocol for Trypsin-assisted Protein Digestion for Proteinsin Solution

A surfactant-assisted in-solution digestion protocol was compared with aurea-assisted protocol and both were used to identify a proteome (set ofproteins) of membrane proteins from mouse heart. Mouse membrane proteinswere digested separately, using urea and surfactant compound 3211,respectively.

Surfactant-Aided Protocol:

Protein from a 50 μg sample of mouse heart membrane extract wasprecipitated with 4 volumes of ice-cold acetone at −80° C. for 20minutes. The precipitated membrane protein mixture was collected bycentrifugation and the protein pellet was washed with 300 μL of coldacetone. The pellet was solubilized in 20 μL of 0.2% surfactant 3211 (in50 mM ammonium bicarbonate), then diluted to a volume of 93.6 μL with 50mM ammonium bicarbonate. After dilution, 1 μL of 0.45M DTT was added andthe sample was incubated for 20 minutes at 56° C. DTT reduction wasfollowed by alkylation with 2 μL of 0.7M iodoacetamide for 15 minutes atroom temperature.

After alkylation, an additional 1 μL of 1% surfactant 3211 was addedfollowed by 3.5 μL of trypsin (0.5 μg/μL in 50 mM ammonium bicarbonate)and the mixture was digested for 3 hours at 37° C.

Urea-Aided Protocol:

Protein from a 50 μg sample of mouse heart membrane extract wasprecipitated with 4 volumes of ice-cold acetone at −80° C. for 20minutes. The precipitated membrane protein mixture was collected bycentrifugation and the protein pellet was washed with 300 μL of coldacetone. The pellet was solubilized in 15 μL of 8M urea, and thendiluted to a volume of 93.6 μL with 50 mM ammonium bicarbonate. Afterdilution, 1 μL of 0.45M DTT was added and the sample was incubated for20 minutes at 56° C. DTT reduction was followed by alkylation with 2 μLof 0.7M iodoacetamide for 15 minutes at room temperature.

After alkylation, 3.5 μL of trypsin (0.5 μg/μL in 50 mM ammoniumbicarbonate) was added and the mixture was digested overnight at 37° C.

Surfactant and Urea-Aided Post Digestion Clean-Up and Analysis.

After digestion, 6 μL of 10% TFA was added and the mixture was incubatedfor 15 minutes at 37° C. Following this acid degradation step the samplewas extracted on a 100 μL C18 OMIX tip (Varian, Inc.) following themanufacturer's instructions, eluting with 20 μL of 70% acetonitrile,0.1% TFA. Each digest mixture was analyzed by 1D nanoLC-MS/MS.

FIG. 4A and FIG. 4B show the total ion chromatogram (TIC) for eachdigestion. As can be observed from the figures, peptide yield and numberof identified peptides were higher in the digest using surfactantcompound 3211. FIG. 5 shows that the surfactant-assisted protocolallowed for ˜70% increase in proteome coverage versus urea (standardsolubilizer for membrane proteins)-assisted protocol.

While 31 proteins were identified in the membrane protein digest withurea, 54 proteins were identified in the membrane protein digest usingsurfactant compound 3211. A higher individual protein coverage was alsoobtained using surfactant compound 3211. The score and coverage forAtp5b was almost double of that obtained using urea. Atp5b protein fromdigest with urea resulted in a score of 384 and 8 peptides (22.5%coverage), while Atp5b protein from digest with compound 3211 resultedin a score of 707 and 15 peptides (42.4% coverage).

It was found that only a portion of identified proteins were common inboth digests, while the remaining digests were specific for eitherdigest, as illustrated in FIG. 5. The use of surfactants of theinvention therefore allow for larger proteome coverage when used inconjunction with a separate urea digest. A digest using a combination ofurea and a surfactant disclosed herein can also be employed. In such aprotocol, each of the proteins identifiable by either reagent, bothurea- and surfactant-specific, can be identified in one digest.

Example 5. Protocol for Trypsin-assisted Protein Digestion for Proteins‘In Gel’

The following is a standard ‘in-gel’ digestion sample preparationprotocol, followed by a description of a sample preparation protocolusing surfactant of the invention.

Materials:

Gel staining solution (for example, with Coomassie Blue); Gel destainingsolution (for example, 40% Ethanol/10% Acetic acid); Highly puredistilled water; 50 mM Ammonium Bicarbonate (AmBic); 25 mMDithiothreitol (DTT); 55 mM Iodoacetamide (IAA); Acetonitrile (ACN);Trypsin; and Trifluoroacetic acid (TFA).

Standard Protocol:

1. Resolve proteins in polyacrylamide gel and stain proteins withStaining solution.

2. Destain the gel with Destaining solution to remove nonspecificallybound stain.

3. Excise protein band of interest, cut the gel slice onto 1 mm³ piecesand transfer into 0.5 or 1.5 mL microcentrifuge polypropylene tube.

4. Wash the pieces with water.

5. Destain the pieces twice with Methanol:50 mM AmBic=1:1 (v/v).

6. Dehydrate for 5 minutes with ACN:50 mM AmBic=1:1 (v/v), then for 30seconds in 100% ACN.

7. Dry in Speed Vac for 5 minutes.

8. Rehydrate in freshly prepared 25 mM DTT and incubate at 56° C. for 20minutes.

9. Discard DTT solution and add freshly prepared 55 mM IAA. Incubate inthe dark at room temperature for 20 minutes.

10. Wash twice with water.

11. Dehydrate for 5 minutes with ACN:50 mM AmBic=1:1 (v/v), then for 30seconds in 100% ACN.

12. Dry in Speed Vac for 5 minutes.

13. Rehydrate for 5 minutes in 20 uL 12 ng/uL Trypsin Gold (in 50 mM ACNM AmBic). Overlay with minimal amount of 50 mM AmBic to cover gelpieces. Incubate overnight at 37° C.

14. Mix the slices with 50 uL 2.5% TFA on Vortex or orbital shaker for15 min. Save the extract.

15. Add 80 uL 70% ACN/5% TFA and mix on Vortex or orbital shaker for 15minutes.

16. Combined both extracts and dry with Speed Vac for 1.5-2 hours.

17. Redissolve peptides in 30 uL 0.1% TFA by mixing on Vortex for 5minutes and clean-up peptides with C18 tips. Analyze with MALDI-TOF.

Sample Preparation with an Enzyme Surfactant as Disclosed Herein.

Materials:

Gel staining solution (for example, with Coomassie Blue); Gel destainingsolution (for example, 40% Ethanol/10% Acetic acid); Highly puredistilled water; 50 mM Ammonium Bicarbonate (AmBic); 25 mMDithiothreitol (DTT); 55 mM Iodoacetamide (IAA); Acetonitrile (ACN);Trypsin; Trifluoroacetic acid (TFA); an enzyme (e.g., trypsin)surfactant A. Surfactant A refers to an acid-labile and/or thermolabilesurfactant as disclosed herein.

Improved Protocol:

1.-12. Steps as above.

13. Rehydrate for 5 minutes in 20 μL 12 ng/μL Trypsin Gold (in 0.025%surfactant A/50 mM ACN M AmBic). Overlay with 30 μL of 0.01% surfactantA/50 mM AmBic to cover gel pieces. Incubate 1 hour at 50° C.

14. Transfer the digestion reaction into a new tube and analyze thedigestion reaction with mass spectrometry.

The surfactant-assisted protocol illustrated in FIG. 6 demonstrates asignificant innovation to in-gel protein digestion. Typical in-geldigestion protocol includes two steps: protein digestion that generallytakes overnight (8-24 hours) and peptide extraction, which typicallyincludes two extractions, including TFA and a TFA/acetonitrile mixture,followed by peptide concentration with 1.5-2 hours of drying in aSpeedVac® concentrator, and reconstitution in a small volume of TFA. Inthe surfactant-assisted protocol, both steps are combined into a singleone hour step leading to dramatic increases in efficiency, includingreduced time and labor required to complete the protocol.

FIG. 7A and FIG. 7B illustrate experimental evidence showing that thesurfactants of the invention extract more peptides, and higher molecularweight peptides, from the gel. The figures show spectra of peptidespresent in a digestion mix, as discussed in the two protocol above. FIG.7A illustrates peptides present in a digestion reaction after overnightdigestion of BSA (50 ng) without the aid of a surfactant of theinvention. FIG. 7B shows the digestion reaction results after one hourdigestion with the aid of a surfactant of the invention. The surfactantdramatically increases peptide recovery, leading to increases in proteincoverage to 36%, up from 8% in a conventional protocol.

Use of surfactant 3211 instead of organic solvent in extraction stepallows one to avoid a time-consuming vacuum drying step (1.5-2 hours)that is required to remove the solvent that, otherwise, interferes withthe peptide clean-up step. In addition, the vacuum drying step can leadto loss of substantial amount of peptides due to strong absorption totube walls. The use of surfactant 3211 ensures high recovery of peptidesby preventing this absorption.

The surfactant offers several improvements over regular protocols forin-gel protein digestion, including improved protein digestion, improvedtrypsin stability, and an innovative surfactant-facilitated peptideextraction as described herein. Combination of these improvements leadto a 2.2 fold improvement in protein coverage and 6.8 fold improvementin Mascot score in experiments conducted using the above protocol.

The surfactants of the invention offer additional innovations to proteindigestion for both ‘in-solution’ and ‘in-gel’ techniques. Earlierdeveloped trypsin surfactants, such as RapiGest™ and PPS SilentSurfactant™, require incubation of protein digestion mixture with acidto degrade surfactant that can otherwise interfere with downstreamapplications, such as liquid chromatography and mass spectrometryanalysis. An important advantage of the surfactants of the invention isthat they degrade by end of the recommended digestion incubation time (3hours) or during preparation of extracted peptides for clean-up step tosuch an extent that no additional degradation is required.

If the surfactant of the invention still remains intact (for example, ifprotein digestion was shorter than 3 hours) and further degradation isrequired, the surfactant can be degraded by elevated temperature as anoption to acid degradation. Currently used products (e.g., RapiGest™surfactant and PPS Silent Surfactant™) require acid degradation. Noevidence that those surfactants can be degraded by elevated temperatureis available. Thermodegradation allows for more flexibility withdownstream treatment of digested peptides. For example, a researcher hasoption to add formic acid instead of TFA to peptide solution anddirectly proceed to peptide separation by liquid chromatography.Thermodegradation can lead to a better recovery of acid labilepost-translational modifications (such as acid labile glycans).Additionally, thermodegradation will be preferred by researchers overacid degradation to avoid the necessity of handling any hazardouschemical material such as strong acids (e.g., TFA) in the protocol.

The protocol can also be aided by use of a urea/surfactant combination.A more diverse population of identified proteins can be observed indigests performed in the presence of urea/surfactant mixtures that inthe presence of either agent alone.

Example 6. Trypsin-Assisted Protein Digestion for Proteins ‘In Gel’

Total membrane protein from mouse heart was resolved inSDS-polyacrylamide gel and a single protein band was digested with aconventional digestion protocol (control) and separately with a compound3211-assisted protocol.

A band of the protein with an approximate MW of 56 kD was excised anddigested with trypsin. The digested samples were analyzed by massspectrometry. FIG. 8A and FIG. 8B show the mass spectra of the twoanalyses; FIG. 8A shows the spectrum obtained from the conventionalovernight protocol without surfactant 3211; FIG. 8B shows the spectrumobtained from the protocol that employed surfactant 3211 and only 1 hourof digestion. Asterisks indicate indentified peptides for beta subunitof mouse ATP synthase (the major protein in 56 kD band).

FIG. 8B shows a dramatic increase in the number, and yield, of peptidesobtained using the surfactant of the invention protocol. The protocolwithout surfactant provided sequence coverage for beta subunit of ATPsynthase of 50% and a MASCOT score of 828, while the surfactant aidedprotocol provided a 75% sequence coverage (a 50% increase) and a MASCOTscore of 920. Use of a surfactant of the invention, such as compound3211, substantially improves mass spectrometry protein identificationand simplifies in-gel digestion protocol by combining lengthy proteindigestion and peptide extraction steps into a single one hour step.

One dimensional (1D) gel techniques are frequently used to fractionatecomplex protein mixtures. A protein mixture is resolved in-gel and thegel line is cut onto gel slices. Each gel slice represents a proteinfraction. The fractions are then in-gel digested and digested peptidesare then identified using LC-MS/MS. Use of the surfactants of theinvention afford significant savings in terms of time and labor inpreparing gel-fractionated proteins for mass spec analysis.

FIG. 9 illustrates how the surfactant-assisted one hour protocolprovides a satisfactory or superior method for identification of complexprotein mixtures in gel. In the experiment, a membrane protein extractfrom mouse heart was resolved in 4-20% SDS-PAGE and the fractioncontaining proteins with approximate MW of 54-56 kD was analyzed withLC-MS/MS. The number of proteins identified after a one hour digestionwith the surfactant was greater than number of proteins identified witha conventional overnight digestion protocol. Additionally, higherprotein coverage was achieved for one third of the identified proteinsin the surfactant-assisted protocol.

Example 7. Trypsin-Assisted Protein Digestion for Proteins ‘In Gel’

A surfactant of the invention can improve in-sodium dodecyl sulfatepolyacrylamide gel protein digestion for matrix-assisted laserdesorption/ionization mass spectrometric peptide mapping. Massspectrometry, in conjunction with genome database searches, is a toolfor the identification of proteins. In proteome analysis, mixtures ofcellular proteins are often separated by sodium dodecyl sulfate (SDS)polyacrylamide gel-based one-dimensional gel electrophoresis (1-DE) ortwo-dimensional gel electrophoresis (2-DE), and in-gel digested by aspecific protease. In-gel protein digestion is an important step forsensitive protein identification by these procedures. Efficient proteindigestion is helpful for obtaining the peptide peaks necessary for clearprotein identification by mass spectrometry.

A significant improvement of protein digestion in SDS polyacrylamidegels using an surfactant of the invention can be obtained. Thesurfactants described herein can also dramatically improve peptideyields by making peptide extraction more efficient (for example, see thetechniques described by Nomura et al., J. Mass Spec. 2004, 39(2),202-207, and specifically page 203 for ‘in-gel’ digestion and extractionof peptides with a surfactant, incorporated by reference herein), and bypreventing absorption of peptides by labware, or by preventing peptideloss due to precipitation. This provides a useful strategy for sensitiveprotein identification by mass spectrometry. The surfactants describedherein also offer a dramatic simplification to in-gel protein digestionby combining lengthy protein digestion and peptide extraction into asingle one hour step.

Example 8. Trypsin-Assisted Protein Digestion for Proteins ‘In Solution’

Standard biochemical techniques used for protein isolation, such asaffinity isolation and density gradient centrifugation, can providenanogram to low-microgram quantities of protein material with asignificant amount of labor and time. The effectiveness of massspectrometry characterization of isolated protein by a “shotgun”approach is often reduced by a lack of effective and efficientin-solution proteolysis protocols for these small quantities ofproteins. Proteolytic peptides can be analyzed by one-dimensional liquidchromatography-tandem mass spectrometry (LC-MS/MS). The effectiveness ofa digestion protocol can be assessed on the basis of, for example, threeparameters: number of peptide identifications, number of proteinidentifications, and sequence coverage. Use of 80% acetonitrile fortrypsin digestions can be advantageous, and is often better thanprotocols employing other solvents and chaotropes in various proteinisolates. A primary advantage of the 80% CH3CN protocol is that it canrequire fewer sample manipulation steps. See for example, the methodsused to denature proteins prior to digestion with trypsin for‘in-solution’ reactions discussed by Hervey et al., J. Proteome Res.2007; 6(8); 3054-3061, in particular, the discussion at page 3055,incorporated by reference herein.

Example 9. Surfactant-Assisted Protein Digestion for Proteins ‘InSolution’

An optimization and comparison of trypsin digestion strategies forpeptide/protein identifications by LC-MS/MS with or without massspectrometry compatible detergents in mixed organic-aqueous and aqueoussystems can be carried out using the acid- and thermolabile compoundsdisclosed herein. It can be shown that adding massspectrometry-compatible surfactants to proteolytic digestion protocolsdramatically increases peptide and protein identifications in complexprotein mixtures by shotgun proteomics. Protein solubilization andproteolytic efficiency can be increased by including massspectrometry-compatible detergents in trypsin digestion buffers.

A modified trypsin digestion protocol incorporating the massspectrometry-compatible surfactants can aid in the identification ofproteins from, for example, pancreatic cell lysates, and can generate agreater number of peptide identifications than trypsin digestion withurea when using LC-MS/MS. Additionally, proteins can be identified bymerging protein identifications from trypsin digestion with various massspectrometry-compatible surfactants. It can also be observed that theuse of mixed aqueous and organic solvent systems can influence proteinidentifications in combinations with different massspectrometry-compatible surfactants.

Peptide mixtures generated from different mass spectrometry-compatiblesurfactants and buffer combinations can show a significant difference inhydrophobicity. Experimental results show that protein digestion schemesincorporating mass spectrometry-compatible surfactants can generatequantitative as well as qualitative changes in observed peptideidentifications, leading to increased protein identifications overalland increased identification of low-abundance proteins. See for example,the techniques described by Chen et al., J. Proteome Res. 2007; 6(7);2529-2538, and in particular at page 2531 for a discussion of theability of acid-labile surfactants to improve proteolysis of complexprotein mixtures with trypsin, incorporated by reference herein.

The surfactants described herein not only improve protein solubilizationand digestion, they also substantially increase peptide yield bypreventing peptide loss due to absorption by labware or precipitation.This three-way benefit makes the surfactants an ideal means forin-solution digestion protocol. Initial evidence indicates that thesurfactants can be also used in combination with urea, a standardsolubilizing agents for in-solution digestion protocol. The surfactantscan retain all of their beneficial properties in the presence of urea.Digestion in urea/surfactant mixture can provide a larger number ofidentified proteins, larger individual protein coverage, and improvedmass spectrometric analysis data than with the use of these the agentsindividually.

Example 10. Cyanogen Bromide Protein Digestion

In protein digestion, cyanogen bromide (CNBr) acts by cleavage of aprotein at methionine residues resulting in a homoserine lactone on thecarboxyl fragment. Cyanogen bromide is useful for analysis of membraneproteins because methionine residues are often located in hydrophobicregions of a protein and cleavage advantageously reduces the size of thehydrophobic fragment, which increases the efficiency and effectivenessof analysis. Cyanogen bromide can be used by itself to produce largefragments, or in series prior to a protease such as trypsin orchymotrypsin. Typical protocols require some type of surfactant to aidin solubilization of the protein mixture of interest before reacting theprotein with an excess of CNBr in the dark for 24 hours. The digestreaction requires low pH so it is often run in 50-80% TFA of HClsolutions. Typical solubilizing agents may need to be removed from thereaction before analysis while excess CNBr is volatized.

The surfactant compounds of formula I-IX can be used to aid in thesolubilization of a membrane protein mixture, followed by adjustment ofthe reaction conditions to low pH, thereby degrading the surfactantprior to addition of CNBr. Following digestion the sample can beanalyzed directly by MALDI with no need to remove the denaturing agentsthat would otherwise interfere in ionization.

Example 11. Deglycosylation Techniques Using Surfactants

Glycosylation is important post-translational protein modificationimplicated in many key cellular mechanisms and disorders. In fact, about60% of human proteins are glycosylated. A protein can be glycosylated atvarious sites with different types of carbohydrates. The function of aprotein is affected in different ways, depending on which site of aprotein is glycosylated, and what type of carbohydrate is attached.Accordingly, glycosylation analysis presents significant challenges, andimproved techniques for conducting the analyses are needed.

Mass spectrometry is a major analytical tool for study of glycosylation.This powerful technique allows for localizing sites of glycosylation andidentifying carbohydrate structures. One significant challenge is thatthe protein and carbohydrate moieties of a glycoprotein should bephysically separated for efficient and effective analysis, i.e., eachmoiety must be analyzed separately to obtain appropriate data. Theseparation of these moieties can be achieved by enzymaticdeglycosylation. Enzymatic deglycosylation is often time-consumingand/or inefficient because glycosylated sites of glycoproteins are oftenobstructed by a protein's structure.

To allow glycosidases easy access to glycosylated sites of a protein,researchers frequently use SDS. SDS greatly improves deglycosylation,however it interferes with mass spectrometric analysis. Researchersstudying cancer, cellular mechanisms, and other biological problemsroutinely treat protein samples with glycosidases prior to massspectrometric analysis. The information commonly sought includes whichsites of a protein of interest carry carbohydrates. These sites can belocalized after deglycosylation because glycosidases leave behind asmall portion of carbohydrates after cleavage. This remaining portionacts as marker of glycosylated site in mass spectrometric analysis. Someresearchers are also interested in analyzing the cleaved carbohydrates.

Surfactants described herein were evaluated as replacements for SDS indeglycolsylation protocols. Ovalbumin (albumin from chicken egg white)was selected as a model protein. Ovalbumin is a 44.3 kD glycoproteinwith 1.4 kD carbohydrate units linked to the protein. Ovalbumindeglycosylation is inefficient under standard conditions, even afterovernight incubation with PNGase F. The ovalbumin was treated withglycosidase PNGase F. It was found that, in the presence of surfactantcompound 3266, PNGase F rapidly removed carbohydrate from ovalbumin.Surfactant compound 3266 is acid-labile and it can be easily degraded,allowing for analysis of the protein and carbohydrate using massspectrometric analysis.

Detailed Protocol:

Ovalbumin (Sigma) (45 μg) was dissolved in 0.025% 3266/5 mM DTT/25 mMammonium bicarbonate (pH˜8) and incubated at 95° C. for 5 minutes. Inthe control reaction, 3266 was replaced with SDS. Deglycosylation wasinitiated by adding 1.5μ units PNGase F (Sigma). After 1 hour and 20minutes of incubation at 37° C., the reactions were terminated byincubation at 95° C. for 5 minutes and an aliquot from each reaction wasresolved with SDS-PAGE. The electrophoresis showed that 44.3 kDglycosylated form of ovalbumin was transformed into a deglycosylated42.9 kD form in both reactions. The analyses indicate that compound 3266is equivalent to the deglycosylation enhancement found using SDS, whileeliminating the deleterious effects of SDS in concomitant spectrometricand chromatographic analysis.

Example 12. Cleavable Surfactants

The following are surfactant compounds of the invention, useful for themethods described herein.

FIGS. 11A-18B show HPLC chromatograms resulting from a control digest(RapiGest™ surfactant) and a surfactant compound of the invention, asnoted below. In each set of chromatograms of FIGS. 11A-18B, the topchromatogram is the control and the bottom chromatogram is the testusing a compound of the invention.

Experimental Conditions: Myoglobin/Trypsin Digest:

Myoglobin from horse heart (typically 25 μs) was digested with trypsin(Promega) at a 50:1 ratio in a volume of 125 μL (typical) of 50 mMAmmonium bicarbonate for 20 minutes at 37° C. with 0.01% RapiGest™surfactant or 0.01% of the indicated compound of the invention (upperand lower chromatograms, respectively). TFA was added to 0.5-2% and themixtures were incubated at 37° C. or 65° C. for 20 or 30 minutes.Degraded surfactant was removed by centrifugation at 14,000×g for 5minutes. HPLC analysis was performed on a 25-50 μL reaction sample usingan HP1050 LC system with an Agilent Sorbax SB-C18 column (3.0×100 mm,3.5 micron) and a gradient of 0.1% TFA (97.5% initial) and Acetonitrile,0.01% TFA (55% final condition followed by a 100% wash step) and a flowrate of 0.75 mL/min. Peptides were detected at 214 and 280 nm.

Experimental Conditions: Bacteriorhodopsin/Chymotrypsin Digest forCompounds 3116-3210, 3213-3223 and 3225-3275:

Bacteriorhodopsin (25 μs) was heated with 0.025% Rapigest™ surfactant orthe indicated compound of the invention for 5 minutes at 95° C., thendigested with chymotrypsin (Sigma) at a 10:1 ratio in 125 μL 50 mMAmmonium bicarbonate for 1 hour at 37° C. After one hour TFA was addedto 0.5-2% and the mixtures were incubated at 37° C. or 65° C. for 20 or30 minutes. Degraded surfactant was removed by centrifugation at14,000×g for 5 minutes. An aliquot of 50-100 μL of digested protein wasanalyzed by HPLC as described above.

Experimental Conditions: Bacteriorhodopsin/Chymotrypsin Digest forCompounds 3211, 3212 and 3224:

Rapigest™ surfactant (control) reaction: As described above.

Compound 3211, 3212, 3224 and Rapigest™ surfactant control for FIG. 13Creactions: Bacteriorhodopsin (25 μs) was solubilized with 0.025%compound of interest for 1-2 minutes at room temperature, then digestedwith chymotrypsin (Sigma) at a 10:1 ratio in 125 μL 50 mM Ammoniumbicarbonate for 1 hour at 37° C. After one hour TFA was added to 0.5-2%and the mixtures were incubated at 37° C. or 65° C. for 20 or 30minutes. Degraded surfactant was removed by centrifugation at 14,000×gfor 5 minutes. An aliquot of 50-100 μL of digested protein was analyzedby HPLC as described above.

For each compound numbered below in Example 12, the rate of enhanceddigestion was determined from the ratio of the surfactant peak area(surfactant # Pk. Area) to the Rapigest™ surfactant peak area (RG Pkarea). Peak area was determined as the sum of all peaks integrated from8-22 minutes. A ratio greater than one indicates more digestion wasobserved for the surfactant reaction than the Rapigest™ surfactantcontrol reaction.

Compound 3116:

Myoglobin/Trypsin digest: 3116 Pk area/RG Pk area=0.6.Bacteriorhodopsin/Chymotrypsin digest: 3116 Pk area/RG Pk area=1.5.

Compound 3116 has an acceptable solubility in aqueous solutions: 0.5% ormore and showed acid-lability: 0.01% 3116 was degraded within 2 hours at37° C. See FIG. 11A and FIG. 11B.

Compound 3186:

Myoglobin/Trypsin digest: 3186 Pk area/RG Pk area=N.D.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3186 for 5minutes before adding chymotrypsin; 3186 Pk area/RG Pk area=0.45.

Compound 3186 shows some solubility in aqueous solution and degradationof a 0.01% solution was complete within 30 minutes at 37° C.

Compound 3189:

Myoglobin/Trypsin digest: 3189 Pk area/RG Pk area=1.1Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3189 beforeadding chymotrypsin; 3189 Pk area/RG Pk area=0.6.

Compound 3189 has a good solubility in aqueous solution; it was easilydissolved to 0.5%. Acid-lability was poor: 0.01% 3189 degraded onlyafter overnight incubation at 37° C.

Compound 3190:

Myoglobin/Trypsin digest: 3190 Pk area/RG Pk area=N.D.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3190 beforeadding chymotrypsin; 3190Pk area/RG Pk area=0.34.

Compound 3190 has a good solubility in aqueous solutions (easilydissolved to 0.5%). Moderate degradation rate: 30 minutes for 0.01%solution, overnight—for 0.1% solution.

Compound: 3192

Myoglobin/Trypsin digest: 3192 Pk area/RG Pk area=1.3Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3192 beforeadding chymotrypsin; 3192 Pk area/RG Pk area=1.1.

Compound 3192 has a good solubility in aqueous solutions (easilydissolved to 0.5%). Degradation rate was very low: 0.01% solutiondegraded only after overnight incubation.

Compound: 3194

Myoglobin/Trypsin digest: 3194 Pk area/RG Pk area=0.72Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3194 beforeadding chymotrypsin; 3194 Pk area/RG Pk area=1.1.

Good solubility in aqueous solutions (easily dissolved to 0.5%). Nodegradation was observed even after overnight incubation.

Compound: 3195

Myoglobin/Trypsin digest: 3195 Pk area/RG Pk area=0.5Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3195 beforeadding chymotrypsin; 3195 Pk area/RG Pk area=N.D.

Moderate solubility in aqueous solutions: not more than 0.2%. Nodegradation was observed even after overnight incubation.

Compound: 3196

Myoglobin/Trypsin digest: 3196 Pk area/RG Pk area=0.65Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3196 beforeadding chymotrypsin; 3196 Pk area/RG Pk area=N.D.

Good solubility in aqueous solutions: easily dissolved to 0.5%. Moderatedegradation rate: 30 minutes for 0.01% solution, overnight—for 0.1%solution.

Compound: 3199

Myoglobin/Trypsin digest: 3199 Pk area/RG Pk area=0.37Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3199 beforeadding chymotrypsin; 3199 Pk area/RG Pk area=N.D.

Good solubility in aqueous solutions: easily dissolved to 0.5%. Nodegradation was observed even after overnight incubation.

Compound: 3200

Myoglobin/Trypsin digest: 3200 Pk area/RG Pk area=0.7Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3200 beforeadding chymotrypsin; 3200 Pk area/RG Pk area=0.8.

Low solubility in aqueous solutions: ˜0.01%. No degradation was observedeven after overnight incubation.

Compound: 3201

Myoglobin/Trypsin digest: 3201 Pk area/RG Pk area=0.3Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3201 beforeadding chymotrypsin; 3201 Pk area/RG Pk area=0.4.

Good solubility in aqueous solutions: easily dissolved to 0.5%.

Compound 3202:

Myoglobin/Trypsin digest: 3202 Pk area/RG Pk area=1.1.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3202 beforeadding chymotrypsin; 3202 Pk area/RG Pk area=0.6.

Somewhat low solubility: ˜0.01%. Degradation rate was variable becauseof lower surfactant solubility: 0.01% 3202 degraded within 30 minutes.See FIG. 12A and FIG. 12B.

Compound: 3203

Myoglobin/Trypsin digest: 3203 Pk area/RG Pk area=N.D.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3203 beforeadding chymotrypsin; 3203 Pk area/RG Pk area=0.4.

Good solubility: easily dissolved to 0.5%. Degradation rate is moderate:0.1% 3203 was degraded after overnight incubation.

Compound: 3204

Myoglobin/Trypsin digest: 3204 Pk area/RG Pk area=0.87.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3204 beforeadding chymotrypsin; 3204 Pk area/RG Pk area=0.55.

Good solubility: easily dissolved to 0.5%. Degradation rate is moderate:0.1% 3203 was degraded after overnight incubation.

Compound: 3205

Myoglobin/Trypsin digest: 3205 Pk area/RG Pk area=0.3.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3205 beforeadding chymotrypsin; 3205 Pk area/RG Pk area=0.45.

Good solubility: easily dissolved to 0.5%. No degradation was observedeven after overnight incubation.

Compound: 3206

Myoglobin/Trypsin digest: 3206 Pk area/RG Pk area=0.26.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3206 beforeadding chymotrypsin; 3206 Pk area/RG Pk area=0.8.

Good solubility: easily dissolved to 0.5%. Degradation rate is moderate:0.1% 3203 was degraded after overnight incubation.

Compound: 3207

Myoglobin/Trypsin digest: 3207 Pk area/RG Pk area=N.D.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3207 beforeadding chymotrypsin; 3207 Pk area/RG Pk area=N.D.

Good solubility: easily dissolved to 0.5%. Degradation rate is low:0.01% 3207 degraded within 1 hour).

Compound: 3209

Myoglobin/Trypsin digest: 3209 Pk area/RG Pk area=2.1.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3209 beforeadding chymotrypsin; 3209 Pk area/RG Pk area=N.D.

Good solubility: easily dissolved to 0.5%. No degradation was observedeven after overnight incubation.

Compound: 3210

Myoglobin/Trypsin digest: 3210 Pk area/RG Pk area=0.3.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3210 beforeadding chymotrypsin; 3210 Pk area/RG Pk area=N.D.

Good solubility: easily dissolved to 0.5%. Degradation rate is moderate:0.1% 3203 was degraded after overnight incubation.

Compound: 3211

Myoglobin/Trypsin digest: 3211 Pk area/RG Pk area=1.3.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with Rapigest™surfactant but not with 3211 before adding chymotrypsin; 3211 Pk area/RGPk area=0.9

Good solubility: easily dissolved to 1% in 50 mM ammonium bicarbonate.Excellent degradation rate: 15 minutes in 0.5% TFA at 37° C. or 5minutes of boiling without TFA. Excellent solubilization properties:rapidly solubilized BR at room temperature. Compound 3211 degrades evenat room temperature at a substantial rate. Also see FIG. 13A, FIG. 13Band FIG. 13C.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate

Surfactant conc. ½-life (hr) 0.0025% 0.5 0.025% 1.1 0.025%/20%AcN 60.025%/40%AcN 52 0.025%/60%AcN >100 0.025%/80%AcN >> 0.10% 4.8 1% 211%/50%AcN >100 >> = compound 100% stable over course of experiment.

Hydrolytic stability was measured by monitoring the change in retentiontime of the intact surfactant by reverse-phase HPLC. The ½-life isdefined as the time required to reach 50% degradation.

Compound: 3212

Myoglobin/Trypsin digest: 3212 Pk area/RG Pk area=1.4.Bacteriorhodopsin/Chymotrypsin digest BR was boiled with Rapigest™surfactant but not with 3212 before adding chymotrypsin; 3212 Pk area/RGPk area=N.D.

Good solubility: easily dissolved to 1% in 50 mM ammonium bicarbonate.Excellent degradation rate: 15 minutes in 0.5% TFA at 37° C. or 5minutes of boiling without TFA. Excellent solubilization properties:rapidly solubilized BR at room temperature. Also see FIG. 14A and FIG.14B.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.0025% 3.7 0.025% 21 0.10% 38 1% >> >> = compound 100% stable overcourse of experiment.

Compound: 3213

Myoglobin/Trypsin digest: 3213 Pk area/RG Pk area=0.8Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3213 beforeadding chymotrypsin; 3213 Pk area/RG Pk area=1.3

Moderate solubility: 0.025% after 10 minutes.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.0025% 62 0.025% 128

Compound: 3214

Myoglobin/Trypsin digest: 3214 Pk area/RG Pk area=N.D.Bacteriorhodopsin/Chymotrypsin digest 10:1 ratio: at 0.1%; BR was boiledwith 3214 before adding chymotrypsin; 3214 Pk area/RG Pk area=0.6

Good solubility in aqueous solutions: easily dissolved to 1%.

Compound: 3215

Myoglobin/Trypsin digest: 3215 Pk area/RG Pk area=0.56.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3215 beforeadding chymotrypsin; 3215 Pk area/RG Pk area=1.2.

Good solubility in aqueous solutions: easily dissolved to 1%.Degradation rate: 0.1% solution degrades after 1 hour incubation in 0.5%TFA. Analysis of compound 3215 indicated moderate thermolability.

Compound: 3216

Myoglobin/Trypsin digest: 3216 area/RG Pk area=N.D.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3216 beforeadding chymotrypsin; 3216 Pk area/RG Pk area=0.7.

Insoluble in aqueous solutions. Solubilized to 10% in 100% DMSO.

Compound: 3218

Myoglobin/Trypsin digest: 3218 Pk area/RG Pk area=0.3.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3218 beforeadding chymotrypsin; 3218 Pk area/RG Pk area=0.65.

Insoluble in aqueous solutions. Solubilized to 10% in DMSO.

Compound: 3219

Myoglobin/Trypsin digest: 3219 Pk area/RG Pk area=N.D.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3219 beforeadding chymotrypsin; 3219 Pk area/RG Pk area=N.D.

Good solubility in aqueous solutions: easily dissolved to 1%.

Compound: 3220

Myoglobin/Trypsin digest: 3220 Pk area/RG Pk area=0.2.Bacteriorhodopsin/Chymotrypsin digest 10:1 ratio: at 0.025%; BR wasboiled with 3220 before adding chymotrypsin; 3220 Pk area/RG Pkarea=0.8.

Good solubility in aqueous solutions: easily dissolved to 1%. Excellentdegradation rate: 0.1% 3220 degraded within 15 minutes in 0.5% TFA at37° C. The surfactant had moderate thermolability.

Compound: 3221

Myoglobin/Trypsin digest: 3221 Pk area/RG Pk area=0.13.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3221 beforeadding chymotrypsin; 3221 Pk area/RG Pk area=0.5.

Good solubility in aqueous solutions: easily dissolved to 0.5%.

Compound: 3223

Myoglobin/Trypsin digest: 3223 Pk area/RG Pk area=1.1.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3223 beforeadding chymotrypsin; 3223 Pk area/RG Pk area=1.1.

Solubility potentially limited in aqueous solutions; 0.5% solution wasmilky and generated a jelly top phase after centrifugation. Degradationrate is low:

Compound: 3224

Myoglobin/Trypsin digest: 3224 Pk area/RG Pk area=1.3.Bacteriorhodopsin/Chymotrypsin digest: 3224 Pk area/RG Pk area=0.74.

Moderate solubility in aqueous solutions: 0.25% solution. The solubilitywas dramatically improved after adding ACN to 20%: 2% solution wasprepared. Excellent solubilization properties. BR was solubilized with3224 at room temperature. Also see FIG. 15A and FIG. 15B.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.0025% 1.3 0.025% 8.3 0.10% 23 0.25% 22

Compound: 3225

Myoglobin/Trypsin digest: 3225 Pk area/RG Pk area=1.1.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3225 beforeadding chymotrypsin; 3225 Pk area/RG Pk area=0.9.

Not soluble in aqueous solutions. It was solubilized in DMSO to 10%.Degradation rate is low: slowly degrades in 0.5% TFA at 37° C. Nothermolability was observed.

Compound: 3228

Myoglobin/Trypsin digest: 3228 Pk area/RG Pk area=1.4.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3228 beforeadding chymotrypsin; 3228 Pk area/RG Pk area=1.2.

Good solubility in aqueous solutions: easily dissolved to 1%.Degradation rate is low: 0.1%. See also FIG. 16A and FIG. 16B.

Compound: 3236

Myoglobin/Trypsin digest: 3236 Pk area/RG Pk area=1.4.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3236 beforeadding chymotrypsin; 3236 Pk area/RG Pk area=0.71.

Good solubilization in aqueous solutions: easily dissolved to 1%.Degradation rate is low: no degradation was observed with 0.1% solutionin 0.5% TFA after 30 minutes of incubation at 37° C. or 5 minutes ofboiling without TFA.

Compound: 3237

Myoglobin/Trypsin digest: 3237 Pk area/RG Pk area=1.4.Bacteriorhodopsin/Chymotrypsin digest: BR was boiled with 3237 beforeadding chymotrypsin; 3237 Pk area/RG Pk area=1.

Moderate solubility: 0.5% solution. Moderate degradation was observedafter 30 minutes incubation in 0.5% TFA at 37° C.

Compound 3266:

Myoglobin/Trypsin digest: 3266 Pk area/RG Pk area=1.2.Bacteriorhodopsin/Chymotrypsin digest: 3266 Pk area/RG Pk area=N.D.

Excellent solubility: instantly dissolved to 2% in 50 mM ammoniumbicarbonate. Acid-labile (0.1% solution degraded in 0.5% TFA within 30minutes) but not thermolabile (remained intact after 5 minutes at 95°C.). Also see FIG. 17A and FIG. 17B.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.025% >> 0.25% >> 1% >> >> = compound 100% stable over course ofexperiment.

Compound: 3267

Myoglobin/Trypsin digest: 3267 Pk area/RG Pk area=0.9.Bacteriorhodopsin/Chymotrypsin digest: BR was solubilized withoutboiling prior to adding chymotrypsin; 3267 Pk area/RG Pk area=N.D.

Excellent solubility: dissolved to 2% in 50 mM ammonium bicarbonate. Didnot degrade in 0.5% TFA or by boiling—acid- and thermolabile.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

1%>>>>=compound 100% stable over course of experiment.

Compound: 3269

Myoglobin/Trypsin digest: 3269 Pk area/RG Pk area=1.Bacteriorhodopsin/Chymotrypsin digest: BR was solubilized withoutboiling prior to adding chymotrypsin; 3269 Pk area/RG Pk area=N.D.

Acceptable solubility: prepared 1% solution.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.03% 7 1% 135

Compound: 3270

Myoglobin/Trypsin digest: 3270 Pk area/RG Pk area=0.8.Bacteriorhodopsin/Chymotrypsin digest: BR was solubilized withoutboiling prior to adding chymotrypsin; 3270 Pk area/RG Pk area=N.D.

Moderate solubility: prepared 0.5% 3270.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.025% 6.5 1% 40

Compound: 3271

Myoglobin/Trypsin digest: 3271 Pk area/RG Pk area=0.9.Bacteriorhodopsin/Chymotrypsin digest: 3271 Pk area/RG Pk area=N.D.

Good solubility (1% solution was easily prepared). Just partiallyacid-labile, thermostable (TLC analysis). Also see FIG. 18A and FIG.18B.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.025% >350 1% >> >> = compound 100% stable over course of experiment.

Compound: 3273

Myoglobin/Trypsin digest: 3273 Pk area/RG Pk area=0.75.Bacteriorhodopsin/Chymotrypsin digest: BR was solubilized withoutboiling prior to adding chymotrypsin; Pk area/RG Pk area=N.D.

Good solubility (1% solution was easily prepared). All the propertiesare similar to 3271. Just partially acid-labile, thermostable

Compound: 3274

Myoglobin/Trypsin digest: 3274 Pk area/RG Pk area=1.Bacteriorhodopsin/Chymotrypsin digest: BR was solubilized withoutboiling prior to adding chymotrypsin; 3274 Pk area/RG Pk area=N.D.

Moderate solubility: prepared 1% solution (at room temperature). Acid-and thermolabile.

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.025% ~6 1% N.D. N.D. = biphasic curve uninterruptible.

Compound: 3275

Myoglobin/Trypsin digest: 3275 Pk area/RG Pk area=0.2.Bacteriorhodopsin/Chymotrypsin digest: BR was solubilized withoutboiling prior to adding chymotrypsin; 3275 Pk area/RG Pk area=N.D.

Acceptable solubility: easily prepared 1% solution. Fully acid- andthermostable at the tested conditions

Hydrolytic Stability in 50 mM Ammonium Bicarbonate Surfactant Conc.½-Life (hr)

0.025% >300 1% >> >> = compound 100% stable over course of experiment

Example 13. Protocol for Trypsin-Assisted Protein Digestion for Proteinsin Solution Analyzed by Off-Line 2D-LC-MS/MS

A surfactant-assisted in-solution digestion was compared with aurea-assisted in-solution digestion and with urea+surfactant assistedin-solution digestion to demonstrate the increase in protein coverageone can obtain by either substituting the surfactant for urea as adenaturant, or by supplementing urea with a surfactant when digesting acomplex mixture. To demonstrate the surfactants efficacy as a solubilantand denaturant, three separate samples of Mouse heart membrane proteinextract were solubilized and digested in separate reactions withsurfactant compound 3211 as denaturant, Urea as denaturant andUrea+surfactant compound 3211 together as denaturant. Each digest wasseparately analyzed by off-line 2D-LC-MS/MS and the protein coveragefrom each condition was compared.

Surfactant-Aided Protocol.

Protein from a 500 μg sample of mouse heart membrane extract wassolubilized in 60 μL of 0.2% surfactant 3211 (in 50 mM ammoniumbicarbonate), then diluted to a volume of 280 μL with 50 mM ammoniumbicarbonate. After dilution, 3 μL of 1M DTT was added and the sample wasincubated for 20 minutes at 56° C. DTT reduction was followed byalkylation with 18 μL 0.5M iodoacetamide for 15 minutes at roomtemperature. After alkylation, an additional 3 μL of 1% surfactant 3211was added followed by 17 μg of trypsin (1.0 μg/μL in 50 mM ammoniumbicarbonate) and the mixture was digested for 3 hours at 37° C.

Urea-Aided Protocol.

Protein from a 500 μg sample of mouse heart membrane extract wassolubilized in 45 μL of 8M urea, and then diluted to a volume of 280 μLwith 50 mM ammonium bicarbonate. After dilution, 3 μL of 1M DTT wasadded and the sample was incubated for 20 minutes at 56° C. DTTreduction was followed by alkylation with 18 μL of 0.5M iodoacetamidefor 15 minutes at room temperature. After alkylation, 17 μg of trypsin(1.0 μg/μL in 50 mM ammonium bicarbonate) was added and the mixture wasdigested overnight at 37° C.

Urea+Surfactant-Aided Protocol.

Protein from a 500 μg sample of mouse heart membrane extract wassolubilized in the mixture of 45 μL 8M Urea/60 μL 0.2% compound 3211,then diluted to a volume of 280 μL with 50 mM ammonium bicarbonate.After dilution, 3 μL of 1M DTT was added and the sample was incubatedfor 20 minutes at 56° C. DTT reduction was followed by alkylation with18 μL 0.5M iodoacetamide for 15 minutes at room temperature. Afteralkylation, an additional 3 μL of 1% surfactant 3211 was added followedby 17 μg of trypsin (1.0 μg/μL in 50 mM ammonium bicarbonate) and themixture was digested for 3 hours at 37° C.

Off-Line 2D-LC-MS/MS Analysis.

Each digestion sample was divided into three aliquots, adjusted to 0.5%TFA and solid phase extracted using a 4 mg Varian Spec SPE tip.Triplicate elutions were combined and evaporated to dryness (speed-vac)and dissolved in ˜100 μl of 0.1% TFA and loaded on to an Agilent Zorbax300-SCX column (3.0×50 mm, 5-micron). Solvents: A=0.03% (v/v) formicacid/5% acetonitrile; B=0.03% (v/v) formic acid/5% acetonitrile/0.5Msodium chloride. After loading peptides were eluted by gradient elutionon an HP1050 HPLC monitored by absorbance at 214 nm, eluted peptideswere fractionated into 1 mL fractions (˜20 fractions per condition).Salt gradient: flow rate=1 mL/min; Initial conditions: 0% B, T=0-8minutes 100% B, T=8-20 minutes 100% B. Each SCX fraction wasconcentrated to dryness then dissolved in 0.1% TFA/5% acetonitrile anddesalted using a 100 μL OMIX SPE column eluting with 100 μL of 70%acetonitrile (0.1% TFA). Eluants were evaporated to dryness andre-suspended in 0.1% TFA/5% acetonitrile for 2nd dimension LC-MS usingan Agilent 1100 series LC/MSD Trap SL spectrometer.

Discussion.

Equal amounts of membrane protein extract were solubilized under threedifferent conditions: with urea, with surfactant, or with aurea/surfactant mix. All the protein mixtures were digested withtrypsin. Proteins solubilized with urea were digested overnight.Proteins solubilized with surfactant or the urea/surfactant mix weredigested for 3 hours. The digests were fractionated on an SCX column(strong cation exchange). Each SCX fraction was analyzed in the seconddimension with reverse phase LC-MS/MS.

As a accepted solubilizing denaturant, urea is commonly used to extendthe coverage of proteomic coverage. In this experiment, 477 differentproteins were identified by using urea as a denaturant. Digests thatincluded a surfactant (with and without urea) extended the proteincoverage by approximately 335 proteins that were not observed in thedigest using urea only. This amounts to a 70% increase in proteincoverage by using the surfactant as both an alternative andcomplimentary solubilizer/denaturant.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1-10. (canceled)
 11. A method for protein digestion comprising contacting a gel, a solid support, or a solution comprising a biomaterial that includes at least one protein, with a digestion reagent and a surfactant compound of formula (I), or a salt thereof, to provide a sample comprising at least one digested protein:

wherein: Q is (C₁-C₆)alkyl, (C₆-C₁₀)aryl, (C₅-C₁₀)heteroaryl, or (C₆-C₁₀)aryl-NH(C₁-C₆)alkyl; Y is O, S, NH, —X—C(═O)—, —C═N—, carbonyl, or —O—C(═Z)—X—; A is aryl, aryl(C₁-C₆)alkyl, heteroaryl, or a direct bond; Z is O or S; X is O, NH, or S; V is C or N; M is H, an alkali metal, or tetra(C₁-C₂₀)alkylammonium; L is —X—C(═Z)—X— or a direct bond; R¹ is (C₄-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₆-C₁₆)aryl, (C₅-C₁₀)heteroaryl, (C₁-C₂₀)alkoxy(C₁-C₂₀)alkyl, (C₁-C₁₂)alkyl(C₆-C₂₀)polyalkoxy, or (C₆-C₂₀)(alkylthio)-(C₁-C₆)alkyl, or absent when V is N; R² and R³ are each independently H or (C₁-C₂₀)alkyl; or R² and R³ together form a 3-8 membered carbocycle ring, or a 3-8 membered heterocyclic ring comprising 1, 2, or 3 N(R^(x)), S, or O; or —V(R²)(R³)-L-R¹ is optionally —O—R¹ when A is aryl; wherein any alkyl, alkenyl, aryl, or heteroaryl, carbocyclic ring, or heterocyclic ring, is optionally substituted with one or more (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₁₀)alkynyl, (C₃-C₁₀)cycloalkyl, (C₁-C₂₀)alkoxy, (C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —CO₂R^(x), —SO₂R^(x), —SO₃R^(x), nitro, amino, N(R^(x))₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₁₆)aryl, (C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, or heterocycle groups; provided that Q is not substituted with CO₂H; and each R^(x) is independently H, (C₁-C₆)alkyl, (C₆-C₁₆)aryl, or (C₁-C₆)alkyl-(C₆-C₁₆)aryl.
 12. The method of claim 11 wherein the biomaterial is in a gel or is bound to a solid support.
 13. The method of claim 11 wherein the biomaterial is in an aqueous solution.
 14. The method claim 11 wherein the digestion reagent comprises one or more of a protease, CNBr, or hydroxylamine.
 15. The method of claim 14 wherein the protease is a serine protease.
 16. The method of claim 15 wherein the serine protease is trypsin or chymotrypsin.
 17. The method of claim 11 further comprising decomposing the surfactant compound after protein digestion, wherein the degrading optionally comprises contacting the surfactant compound with an acidic solution, heating the surfactant compound, or a combination thereof.
 18. The method of claim 11 wherein the surfactant compound self-hydrolyses in solution after protein digestion.
 19. The method of claim 11 further comprising isolating the one or more digested proteins.
 20. The method of claim 11 further comprising analyzing the digested proteins, wherein the digested proteins are analyzed by mass spectrometry, liquid chromatography, gel electrophoresis, or a combination thereof. 21-31. (canceled)
 32. The method of claim 11 wherein Q is (C₁-C₆)alkyl, phenyl, or phenyl-NH(C₁-C₆)alkyl.
 33. The method of claim 11 wherein Y is —O—C(═Z)—X—, X is NH, and Z is O.
 34. The method of claim 11 wherein A is a direct bond.
 35. The method of claim 11 wherein M is H or Na.
 36. The method of claim 11 wherein V is C and L is a direct bond.
 37. The method of claim 11 wherein R¹ is (C₄-C₂₀)alkyl substituted with a (C₆-C₁₀)aryl or heteroaryl group.
 38. The method of claim 37 wherein R¹ is (C₄-C₂₀)alkyl substituted with a 2-furanyl group or a 4-methoxyphenyl group.
 39. The method of claim 11 wherein R² and R³ are each (C₁-C₆)alkyl.
 40. The method of claim 11 wherein the surfactant compound of formula (I) is

or a salt thereof. 